Variant CD3-Binding Domains and Their Use in Combination Therapies for the Treatment of Disease

ABSTRACT

The present invention is directed to DA×CD3 Binding Molecules comprising a vCD3-Binding Domain, which comprises a CDR H 1 Domain, a CDR H 2 Domain, a CDR H 3 Domain, a CDR L 1 Domain, a CDR L 2 Domain, and a CDR L 3 Domain, at least one of which differs in amino acid sequence from the amino acid sequence of the corresponding CDR of a rCD3-Binding Domain, wherein the DA×CD3 Binding Molecule comprising such vCD3-Binding Domain exhibits an altered affinity for CD3, relative to a DA×CD3 Binding Molecule comprising such rCD3-Binding Domain. The invention particularly concerns to such DA×CD3 Binding Molecules comprising a vCD3-Binding Domain which exhibit reduced affinity for CD3 and are capable of mediating redirected killing of target cells expressing a DA and exhibit lower levels of cytokine release relative to a DA×CD3 Binding Molecule comprising a rCD3-Binding Domain. The invention particularly concerns the use of DA×CD3 Binding Molecules comprising a vCD3-Binding Domain in the treatment of cancer and pathogen-associated diseases. The present invention is also directed to pharmaceutical compositions that comprise such molecule(s).

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation application of U.S. application Ser.No. 16/966,960, filed Aug. 3, 2020, which is a 35 U.S.C. 371 nationalphase patent application of International Application No.PCT/US2019/017772, filed Feb. 13, 2019, which claims the priority toU.S. Patent Application Ser. Nos. 62/631,043 (filed on Feb. 15, 2018),and 62/738,632 (filed on Sep. 28, 2018), each of which applications areherein incorporated by reference in their entirety.

REFERENCE TO SEQUENCE LISTING

This application includes one or more Sequence Listings pursuant to 37C.F.R. 1.821 et seq., which are disclosed in computer-readable media(file name: 0260-0012US2_SL.xml, created on May 15, 2023, and having asize of 317 KB), which file is incorporated herein in its entirety.

FIELD OF THE INVENTION

The present invention is directed to multispecific Binding Molecules(e.g., a bispecific antibody, a diabody, a bispecific scFv, a trivalentmolecule, a TandAb®, a BiTE® etc.) comprising a CD3-Binding Domaincapable of binding an epitope of CD3 and also a Disease Antigen-BindingDomain capable of binding an epitope of a Disease Antigen (“DA”) (e.g.,a “DA×CD3 Binding Molecule”). The invention particularly concerns suchDA×CD3 Binding Molecules comprising a variant CD3-Binding Domain(“vCD3-Binding Domain”), which comprises a CDR_(H)1 Domain, a CDR_(H)2Domain, a CDR_(H)3 Domain, a CDR_(L)1 Domain, a CDR_(L)2 Domain, and aCDR_(L)3 Domain, at least one of which differs in amino acid sequencefrom the amino acid sequence of the corresponding CDR of a referenceCD3-Binding Domain (“rCD3-Binding Domain”), and wherein the DA×CD3Binding Molecule comprising such vCD3-Binding Domain exhibits an alteredaffinity for CD3, relative to a DA×CD3 Binding Molecule comprising suchrCD3-Binding Domain. The invention particularly concerns to such DA×CD3Binding Molecules comprising a vCD3-Binding Domain which exhibit reducedaffinity for CD3 and are capable of mediating redirected killing oftarget cells expressing a DA and exhibit lower levels of cytokinerelease relative to a DA×CD3 Binding Molecule comprising a rCD3-BindingDomain. The invention particularly concerns the use of DA×CD3 BindingMolecules comprising a vCD3-Binding Domain in the treatment of cancerand pathogen-associated diseases. The present invention is also directedto pharmaceutical compositions that comprise such molecule(s).

BACKGROUND OF THE INVENTION I. The Mammalian Immune System

The mammalian immune system serves as a defense against a variety ofconditions, including, e.g., injury, infection and neoplasia. Theefficiency with which humans and other mammals develop an immunologicalresponse to pathogens, foreign substances and cancer antigens rests ontwo characteristics: the exquisite specificity of the immune responsefor antigen recognition, and the immunological memory that allows forfaster and more vigorous responses upon re-activation with the sameantigen (Portolés, P. et al. (2009) “The TCR/CD3 Complex: Opening theGate to Successful Vaccination,” Current Pharmaceutical Design15:3290-3300; Guy, C. S. et al. (2009) “Organization of Proximal SignalInitiation at the TCR:CD3 Complex,” Immunol Rev. 232(1):7-21; Topalian,S. L. et al. (2015) “Immune Checkpoint Blockade: A Common DenominatorApproach to Cancer Therapy,” Cancer Cell 27:450-461).

In healthy individuals, the immune system is in a quiescent state,inhibited by a repertoire of diverse inhibitory receptors and receptorligands. Upon recognition of a cancer antigen, microbial pathogen, or anallergen, an array of activating receptors and receptor ligands aretriggered to induce the activation of the immune system. Such activationleads to the activation of macrophages, Natural Killer (NK) cells andantigen-specific, cytotoxic, T-cells, and promotes the release ofvarious cytokines, all of which act to counter the perceived threat tothe health of the subject (Dong, C. et al. (2003) “Immune Regulation byNovel Costimulatory Molecules,” Immunolog. Res. 28(1):39-48; Viglietta,V. et al. (2007) “Modulating Co-Stimulation,” Neurotherapeutics4:666-675; Korman, A. J. et al. (2007) “Checkpoint Blockade in CancerImmunotherapy,” Adv. Immunol. 90:297-339). The immune system is capableof returning to its normal quiescent state when the countervailinginhibitory immune signals outweigh the activating immune signals.

Thus, the disease state of cancer (and indeed the disease states ofinfectious diseases) may be considered to reflect a failure toadequately activate a subject's immune system. Such failure may reflectan inadequate presentation of activating immune signals, or it mayreflect an inadequate ability to alleviate inhibitory immune signals inthe subject. In some instances, researchers have determined that cancercells can co-opt the immune system to evade being detected by the immunesystem (Topalian, S. L. et al. (2015) “Immune Checkpoint Blockade: ACommon Denominator Approach to Cancer Therapy,” Cancer Cell 27:450-461).

The mammalian immune system is mediated by two separate but interrelatedsystems: the humoral immune system and the cellular immune system.Generally speaking, the humoral system is mediated by soluble molecules(antibodies or immunoglobulins) produced by B Cells. Such molecules havethe ability to combine with and neutralize antigens that have beenrecognized as being foreign to the body. The cellular immune systeminvolves the mobilization of certain cells, termed “T-cells,” that servea variety of therapeutic roles. T-cells are lymphocytes that mature inthe thymus and circulate between the tissues, lymphatic system and thecirculatory system. In response to the presence and recognition offoreign structures (antigens), T-cells become “activated” to initiate animmune response. In many instances, these foreign antigens are expressedon host cells as a result of neoplasia or infection. Although T-cells donot themselves secrete antibodies, they are usually required forantibody secretion by the second class of lymphocytes, “B Cells” (whichderive from bone marrow). Critically, T-cells exhibit extraordinaryimmunological specificity so as to be capable of discerning one antigenfrom another).

Two interactions are required for T-cell activation (Viglietta, V. etal. (2007) “Modulating Co-Stimulation,” Neurotherapeutics 4:666-675;Korman, A. J. et al. (2007) “Checkpoint Blockade in CancerImmunotherapy,” Adv. Immunol. 90:297-339). In the first interaction, acell must display the relevant target antigen bound to a cell's Class Ior Class II Major Histocompatibility Complex (“MHC”) so that it can bindthe T-cell Receptor (“TCR”) of a naïve T lymphocyte. Although almost allcell types can serve as antigen-presenting cells, some cells, such asmacrophages, B cells, and dendritic cells, specialize in presentingforeign antigens and are “professional” “Antigen-Presenting Cells.”Immunologic detection of antigen bound to an Antigen-Presenting Cell'sMHC I molecules leads to the production of cytotoxic T-cells.Immunologic detection of antigen bound to an Antigen-Presenting Cell'sMHC II molecules leads to the production of cytotoxic T-cells. In thesecond interaction, a ligand of the Antigen-Presenting Cell must bind aco-receptor of the T-cell (Dong, C. et al. (2003) “Immune Regulation byNovel Costimulatory Molecules,” Immunolog. Res. 28(1):39-48; Lindley, P.S. et al. (2009) “The Clinical Utility Of Inhibiting CD28-MediatedCostimulation,” Immunol. Rev. 229:307-321). T-cells experiencing bothstimulatory signals are then capable of responding to cytokines (such asInterleukin-2 and Interleukin-12).

In the absence of both co-stimulatory signals during TCR engagement,T-cells enter a functionally unresponsive state, referred to as clonalanergy (Khawli, L. A. et al. (2008) “Cytokine, Chemokine, andCo-Stimulatory Fusion Proteins for the Immunotherapy of Solid Tumors,”Exp. Pharmacol. 181:291-328). In pathologic states, T-cells are the keyplayers of various organ-specific autoimmune diseases, such as type Idiabetes, rheumatoid arthritis, and multiple sclerosis (Dong, C. et al.(2003) “Immune Regulation by Novel Costimulatory Molecules,” Immunolog.Res. 28(1):39-48).

This immune “checkpoint” pathway is important in maintainingself-tolerance (i.e., in preventing a subject from mounting an immunesystem attack against his/her own cells (an “autoimmune” reaction) andin limiting collateral tissue damage during anti-microbial oranti-allergic immune responses. Where contact of a T-cell results in thegeneration of only one of two required signals, the T-cell does notbecome activated and an adaptive immune response does not occur. The“two signal” mechanism of T-cell activation thus provides a way for theimmune system to avoid undesired responses, such as responses toself-antigens that would otherwise result in an immune system attackagainst a subject's own cells (an “autoimmune” reaction).

II. Cell Surface Molecules of the Cellular Immune System

A. CD3, CD4 and CD8

The cells of the immune system are characterized by their expression ofspecialized glycoprotein cell surface molecules. Interactions betweensuch molecules and molecules of other cells triggers, maintains ordampens the immune response. In particular, all T-cells arecharacterized by their expression of CD3. CD3 is a T-cell co-receptorcomposed of four distinct chains (Wucherpfennig, K. W. et al. (2010)“Structural Biology Of The T-Cell Receptor: Insights into ReceptorAssembly, Ligand Recognition, And Initiation of Signaling,” Cold SpringHarb. Perspect. Biol. 2(4):a005140; pages 1-14; Chetty, R. et al. (1994)“CD3: Structure, Function, And Role Of Immunostaining In ClinicalPractice,” J. Pathol. 173(4):303-307; Guy, C. S. et al. (2009)“Organization Of Proximal Signal Initiation At The TCR:CD3 Complex,”Immunol. Rev. 232(1):7-21).

In mammals, the complex contains a CD37 chain, a CD36 chain, and twoCD3ε chains. These chains associate with the TCR in order to generate anactivation signal in T lymphocytes (Smith-Garvin, J. E. et al. (2009) “TCell Activation,” Annu. Rev. Immunol. 27:591-619). In the absence ofCD3, TCRs do not assemble properly and are degraded (Thomas, S. et al.(2010) “Molecular Immunology Lessons From Therapeutic T-Cell ReceptorGene Transfer,” Immunology 129(2):170-177). CD3 is found bound to themembranes of all mature T-cells, and in virtually no other cell type(see, Janeway, C. A. et al. (2005) In: IMMUNOBIOLOGY: THE IMMUNE SYSTEMIN HEALTH AND DISEASE,” 6th ed. Garland Science Publishing, NY, pp.214-216; Sun, Z. J. et al. (2001) “Mechanisms Contributing To T CellReceptor Signaling And Assembly Revealed By The Solution Structure Of AnEctodomain Fragment Of The CD3ε:γ Heterodimer,” Cell 105(7):913-923;Kuhns, M. S. et al. (2006) “Deconstructing The Form And Function Of TheTCR/CD3 Complex,” Immunity. 2006 February; 24(2):133-139).

The invariant CD3ε signaling component of the TCR complex on T-cells,has been used as a target to force the formation of an immunologicalsynapse between T-cells and cancer cells. Co-engagement of CD3 and thetumor antigen activates the T-cells, triggering lysis of cancer cellsexpressing the tumor antigen (Baeuerle et al. (2011) “Bispecific T-cellEngager For Cancer Therapy,” In: BISPECIFIC ANTIBODIES, Kontermann, R.E. (Ed.) Springer-Verlag; 2011:273-287). This approach allows bispecificantibodies to interact globally with the T-cell compartment with highspecificity for cancer cells and is widely applicable to a broad arrayof cell surface tumor antigens and has also been implemented to targetpathogen-infected cells (see, e.g., Sloan et al. (2015) “Targeting HIVReservoir in Infected CD4 T Cells by Dual-Affinity Re-targetingMolecules (DARTs) that Bind HIV Envelope and Recruit Cytotoxic T Cells,”PLoS Pathog 11(11): e1005233. doi:10.1371/journal.ppat.1005233; WO2014/159940; and WO 2016/054101).

A first subset of T-cells, known as “helper T-cells,” is characterizedby the expression of the CD4 (i.e., they are “CD4⁺” as well as CD3⁺).CD4⁺ T-cells are the essential organizers of most mammalian immune andautoimmune responses (Dong, C. et al. (2003) “Immune Regulation by NovelCostimulatory Molecules,” Immunolog. Res. 28(1):39-48). The activationof CD4⁺ T-cells has been found to be mediated through co-stimulatoryinteractions between an antigen:Major histocompatibility Class II (MHCII) molecule complex that is arrayed on the surface of anAntigen-Presenting Cell (such as a B-Cell, a macrophage or a dendriticcell) and a complex of two molecules, the TCR and a CD3 cell surfacereceptor ligand, both of which are arrayed on the surface of a naïveCD4⁺ T-cell. Activated T helper cells are capable of proliferating intoTh1 cells that are capable of mediating an inflammatory response to thetarget cell.

A second subset of T-cells, known as “cytotoxic T-cells,” arecharacterized by the expression of CD8 (i.e., they are “CD8⁺” as well asCD3⁺). CD8 is a T-cell co-receptor composed of two distinct chains(Leahy, D. J. (1995) “A Structural View of CD4 and CD8,” FASEB J.9:17-25) that is expressed on cytotoxic T-cells. The activation of CD8⁺T-cells has been found to be mediated through co-stimulatoryinteractions between an antigen:major histocompatibility class I (MHC I)molecule complex that is arrayed on the surface of a target cell and acomplex of CD8 and the T-cell Receptor, that are arrayed on surface ofthe CD8⁺ T-cell ((Gao, G. et al. (2000) “Molecular Interactions OfCoreceptor CD8 And MHC Class I: The Molecular Basis For FunctionalCoordination With The T-Cell Receptor,” Immunol. Today 21:630-636).Unlike major histocompatibility class II (MHC II) molecules, which areexpressed by only certain immune system cells, MHC I molecules are verywidely expressed. Thus, cytotoxic T-cells are capable of binding a widevariety of cell types. Activated cytotoxic T-cells mediate cell killingthrough their release of the cytotoxins perforin, granzymes, andgranulysin. Through the action of perforin, granzymes enter thecytoplasm of the target cell and their serine protease function triggersthe caspase cascade, which is a series of cysteine proteases thateventually lead to apoptosis (programmed cell death) of targeted cells.

B. The T-Cell Receptor (“TCR”)

The T-cell Receptor (“TCR”) is natively expressed by CD4+ or CD8+T-cells, and permits such cells to recognize antigenic peptides that arebound and presented by class I or class II MHC proteins ofantigen-presenting cells. Recognition of a pMHC (peptide-MHC) complex bya TCR initiates the propagation of a cellular immune response that leadsto the production of cytokines and the lysis of the Antigen-PresentingCell (see, e.g., Armstrong, K. M. et al. (2008) “Conformational ChangesAnd Flexibility In T-Cell Receptor Recognition Of Peptide-MHCComplexes,” Biochem. J. 415(Pt 2):183-196; Willemsen, R. (2008)“Selection Of Human Antibody Fragments Directed Against Tumor T-CellEpitopes For Adoptive T-Cell Therapy,” Cytometry A. 73(11):1093-1099;Beier, K. C. et al. (2007) “Master Switches Of T-Cell Activation AndDifferentiation,” Eur. Respir. J. 29:804-812; Mallone, R. et al. (2005)“Targeting T Lymphocytes For Immune Monitoring And Intervention InAutoimmune Diabetes,” Am. J. Ther. 12(6):534-550). CD3 is the receptorthat binds to the TCR (Thomas, S. et al. (2010) “Molecular ImmunologyLessons From Therapeutic T-Cell Receptor Gene Transfer,” Immunology129(2):170-177; Guy, C. S. et al. (2009) “Organization Of ProximalSignal Initiation At The TCR: CD3 Complex,” Immunol. Rev. 232(1):7-21;St. Clair, E. W. (Epub 2009 Oct. 12) “Novel Targeted Therapies ForAutoimmunity,” Curr. Opin. Immunol. 21(6):648-657; Baeuerle, P. A. etal. (Epub 2009 Jun. 9) “Bispecific T-Cell Engaging Antibodies For CancerTherapy,” Cancer Res. 69(12):4941-4944; Smith-Garvin, J. E. et al.(2009) “T Cell Activation,” Annu. Rev. Immunol. 27:591-619; Renders, L.et al. (2003) “Engineered CD3 Antibodies For Immunosuppression,” Clin.Exp. Immunol. 133(3):307-309).

The TCR and CD3 complex, along with the CD3 ζ chain zeta chain (alsoknown as T-Cell receptor T3 zeta chain or CD247) comprise the “TCRcomplex” (van der Merwe, P. A. etc. (epub Dec. 3, 2010) “Mechanisms ForT Cell Receptor Triggering,” Nat. Rev. Immunol. 11:47-55; Wucherpfennig,K. W. et al. (2010) “Structural Biology of the T Cell Receptor: Insightsinto Receptor Assembly, Ligand Recognition, and Initiation ofSignaling,” Cold Spring Harb. Perspect. Biol. 2:a005140). The complex isparticularly significant since it contains a large number (ten) ofimmunoreceptor tyrosine-based activation motifs (ITAMs).

Multispecific molecules comprising a CD3 Binding Domain and a bindingdomain specific for a Disease Antigen (“DA”) expressed on a target cellare capable of mediating redirected T-cell killing of such target cells.However, due to the affinity of such molecules for CD3, such moleculesmay be too potent, so as to exhibit undesirable cytokine release fromthe stimulated T-cells. Thus, despite prior advances in identifying themolecules involved in mammalian immune responses, a need remains forimproved therapies for treating cancers and infectious diseases. Thepresent invention provides a panel of variant CD3-Binding Domains havinga range of binding kinetics, which may be used to modulate the cellkilling and/or cytokine release activities of such multispecificmolecules to enhance the therapeutic window. The present invention isdirected to this and other goals.

SUMMARY OF THE INVENTION

The present invention is directed to multispecific Binding Molecules(e.g., a bispecific antibody, a diabody, a bispecific scFv, a trivalentmolecule, a TandAb®, a BiTE® etc.) comprising a CD3-Binding Domaincapable of binding an epitope of CD3 and also a Disease Antigen-BindingDomain capable of binding an epitope of a Disease Antigen (“DA”) (e.g.,a “DA×CD3 Binding Molecule”). The invention particularly concerns suchDA×CD3 Binding Molecules comprising a variant CD3-Binding Domain(“vCD3-Binding Domain”), which comprises a CDR_(H)1 Domain, a CDR_(H)2Domain, a CDR_(H)3 Domain, a CDR_(L)1 Domain, a CDR_(L)2 Domain, and aCDR_(L)3 Domain, at least one of which differs in amino acid sequencefrom the amino acid sequence of the corresponding CDR of a referenceCD3-Binding Domain (“rCD3-Binding Domain”), and wherein the DA×CD3Binding Molecule comprising such vCD3-Binding Domain exhibits an alteredaffinity for CD3, relative to a DA×CD3 Binding Molecule comprising suchrCD3-Binding Domain. The invention particularly concerns to such DA×CD3Binding Molecules comprising a vCD3-Binding Domain which exhibit reducedaffinity for CD3 and are capable of mediating redirected killing oftarget cells expressing a DA and exhibit lower levels of cytokinerelease relative to a DA×CD3 Binding Molecule comprising a rCD3-BindingDomain. The invention particularly concerns the use of DA×CD3 BindingMolecules comprising a vCD3-Binding Domain in the treatment of cancerand pathogen-associated diseases. The present invention is also directedto pharmaceutical compositions that comprise such molecule(s).

In detail, the invention provides a DA×CD3 Binding Molecule comprising aCD3-Binding Domain capable of binding an epitope of CD3 and a DiseaseAntigen-Binding Domain capable of binding an epitope of a DiseaseAntigen, wherein the CD3-Binding Domain comprises:

-   -   (I) (A) a CDR_(H)1 Domain comprising an amino acid sequence        selected from the group consisting of SEQ ID NO:99, SEQ ID        NO:91, SEQ ID NO:93, SEQ ID NO:95 and SEQ ID NO:97;        -   (B) a CDR_(H)2 Domain comprising the amino acid sequence of            SEQ ID NO:58;        -   (C) a CDR_(H)3 Domain comprising the amino acid sequence of            SEQ ID NO:59;        -   (D) a CDR_(L)1 Domain comprising the amino acid sequence of            SEQ ID NO:60;        -   (E) a CDR_(L)2 Domain comprising the amino acid sequence of            SEQ ID NO:61; and        -   (F) a CDR_(L)3 Domain comprising the amino acid sequence of            SEQ ID NO:62; or    -   (II) (A) a CDR_(H)1 Domain comprising the amino acid sequence of        SEQ ID NO:57;        -   (B) a CDR_(H)2 Domain comprising the amino acid sequence of            SEQ ID NO:58;        -   (C) a CDR_(H)3 Domain comprising an amino acid sequence            selected from the group consisting of SEQ ID NO:69, SEQ ID            NO:71, SEQ ID NO:73, SEQ ID NO:75, SEQ ID NO:77, SEQ ID            NO:79, SEQ ID NO:81, SEQ ID NO:83, SEQ ID NO:85, SEQ ID            NO:87, SEQ ID NO:89, SEQ ID NO:101, SEQ ID NO:103, SEQ ID            NO:105 and SEQ ID NO:107;        -   (D) a CDR_(L)1 Domain comprising the amino acid sequence of            SEQ ID NO:60;        -   (E) a CDR_(L)2 Domain comprising the amino acid sequence of            SEQ ID NO:61; and        -   (F) a CDR_(L)3 Domain comprising the amino acid sequence of            SEQ ID NO:62; or    -   (III) (A) a CDR_(H)1 Domain comprising the amino acid sequence        of SEQ ID NO:57;        -   (B) a CDR_(H)2 Domain comprising the amino acid sequence of            SEQ ID NO:58;        -   (C) a CDR_(H)3 Domain comprising the amino acid sequence of            SEQ ID NO:59;        -   (D) a CDR_(L)1 Domain comprising the amino acid sequence of            SEQ ID NO:60;        -   (E) a CDR_(L)2 Domain comprising the amino acid sequence of            SEQ ID NO:61; and        -   (F) a CDR_(L)3 Domain comprising an amino acid sequence            selected from the group consisting of SEQ ID NO:109 or SEQ            ID NO:111; or    -   (IV) (A) a CDR_(H)1 Domain comprising the amino acid sequence of        SEQ ID NO:57;        -   (B) a CDR_(H)2 Domain comprising the amino acid sequence of            SEQ ID NO:58;        -   (C) a CDR_(H)3 Domain comprising the amino acid sequence of            SEQ ID NO:59;        -   (D) a CDR_(L)1 Domain comprising the amino acid sequence of            SEQ ID NO:60;        -   (E) a CDR_(L)2 Domain comprising an amino acid sequence            selected from the group consisting of SEQ ID NO:113 and SEQ            ID NO:115; and        -   (F) a CDR_(L)3 Domain comprising the amino acid sequence of            SEQ ID NO:62.

The invention additionally concerns the embodiment of such DA×CD3Binding Molecule, wherein the CD3-Binding Domain comprises:

-   -   (I) (A) a VL Domain comprising the amino acid sequence of SEQ ID        NO:56;        -   (B) a VH Domain comprising an amino acid sequence selected            from the group consisting of SEQ ID NO:98, SEQ ID NO:68, SEQ            ID NO:70, SEQ ID NO:72, SEQ ID NO:74, SEQ ID NO:76, SEQ ID            NO:78, SEQ ID NO:80, SEQ ID NO:82, SEQ ID NO:84, SEQ ID            NO:86, SEQ ID NO:88, SEQ ID NO:90, SEQ ID NO: 92, SEQ ID            NO:94, SEQ ID NO:96, SEQ ID NO:100, SEQ ID NO:102, SEQ ID            NO:104 and SEQ ID NO:106; or    -   (II) (A) a VL Domain comprising an amino acid sequence selected        from the group consisting of SEQ ID NO:108, SEQ ID NO:110, SEQ        ID NO:112; and SEQ ID NO:114;        -   (B) a VH Domain comprising an amino acid sequence of SEQ ID            NO:55.

The invention additionally concerns the embodiment of such DA×CD3Binding Molecule, wherein the DA×CD3 Binding Molecule is a bispecificantibody, a bispecific diabody, a bispecific scFv, a bispecific TandAb,or a trivalent binding molecule.

The invention additionally concerns the embodiment of such DA×CD3Binding Molecule, wherein the DA×CD3 Binding Molecule is capable ofbinding more than one Disease Antigen and/or a different cell surfacemolecule of an effector cell. Particularly, wherein the different cellsurface molecule of an effector cell is CD2, CD8, CD16, TCR, NKp46, orNKG2D.

The invention additionally concerns the embodiment of such DA×CD3Binding Molecule, wherein the Disease Antigen is a Cancer Antigen, or aPathogen-Associated Antigen.

The invention additionally concerns the embodiment of such DA×CD3Binding Molecule, wherein the Cancer Antigen is selected from the groupconsisting of the Cancer Antigens: 19.9, 4.2, ADAM-9, AH6, ALCAM, B1,B7-H3, BAGE, beta-catenin, blood group ALe^(b)/Le^(y), Burkitt'slymphoma antigen-38.13, C14, CA125, Carboxypeptidase M, CD5, CD19, CD20,CD22, CD23, CD25, CD27, CD28, CD33, CD36, CD40/CD154, CD45, CD56, CD46,CD52, CD56, CD79a/CD79b, CD103, CD123, CD317, CDK4, CEA,CEACAM5/CEACAM6, C017-1A, CO-43, CO-514, CTA-1, CTLA-4, Cytokeratin 8,D1.1, D₁56-22, DR5, E₁ series, EGFR, an Ephrin receptor, EphA2, Erb,GAGE, a GD2/GD3/GM2 ganglioside, GICA 19-9, gp100, Gp37, gp75, gpA33,HER2/neu, HMFG, Human Papillomavirus-E6/Human Papillomavirus-E7,HMW-MAA, I antigen, IL13Rα2, Integrin β6, JAM-3, KID3, KID31, KS 1/4pan-carcinoma antigen, L6, L20, LEA, LUCA-2, M1:22:25:8, M18, M39, MAGE,MART, mesothelin, MUC-1, MUM-1, Myl, N-acetylglucosaminyltransferase,neoglycoprotein, NS-10, OFA-1, OFA-2, Oncostatin M, p15, p97, PEM, PEMA,PIPA, PSA, PSMA, prostatic acid phosphate, R₂₄, ROR1, a sphingolipid,SSEA-1, SSEA-3, SSEA-4, sTn, the T-cell receptor derived peptide, T₅A₇,TAG-72, TL5, TNF-receptor, TNF-7 receptor, TRA-1-85, a TransferrinReceptor, 5T4, TSTA, VEGF, a VEGF Receptor, VEP8, VEP9, VIM-D5, and Yhapten, Le^(y).

The invention particularly concerns the embodiment of such DA×CD3Binding Molecule, wherein the Cancer Antigen is B7-H3, CEACAM5/CEACAM6,EGRF, EphA2, gpA33, HER2/neu, VEGF, 5T4, IL13Rα2, CD123, CD19, or ROR1.

The invention additionally concerns the embodiment of such DA×CD3Binding Molecule, wherein the Pathogen-Associated Antigen is selectedfrom the group consisting of the Pathogen-Associated Antigens: HerpesSimplex Virus infected cell protein (ICP)47, Herpes Simplex Virus gD,Epstein-Barr Virus LMP-1, Epstein-Barr Virus LMP-2A, Epstein-Barr VirusLMP-2B, Human Immunodeficiency Virus gp160, Human Immunodeficiency Virusgp120, Human Immunodeficiency Virus gp41, Human Papillomavirus E6, HumanPapillomavirus E7, human T-cell leukemia virus gp64, human T-cellleukemia virus gp46, and human T-cell leukemia virus gp21.

The invention additionally concerns the embodiment of such DA×CD3Binding Molecule, wherein the DA×CD3 Binding Molecule comprises: a firstpolypeptide chain and a second polypeptide chain, covalently bonded toone another, wherein:

-   -   (A) the first polypeptide chain comprises, in the N-terminal to        C-terminal direction:        -   (i) a Domain 1, comprising:            -   (1) a sub-Domain (1A), which comprises a VL Domain of a                monoclonal antibody capable of binding to the epitope of                a Disease Antigen (VL_(DA)); and            -   (2) a sub-Domain (1B), which comprises a VH Domain of a                monoclonal antibody capable of binding to the epitope of                CD3 (VH_(CD3));            -   wherein the sub-Domains 1A and 1B are separated from one                another by a peptide Linker; and        -   (ii) a Domain 2, wherein the Domain 2 is a            Heterodimer-Promoting Domain;    -   (B) the second polypeptide chain comprises, in the N-terminal to        C-terminal direction:        -   (i) a Domain 1, comprising            -   (1) a sub-Domain (1A), which comprises a VL Domain of                the monoclonal antibody capable of binding to the                epitope of CD3 (VL_(CD3)); and            -   (2) a sub-Domain (1B), which comprises a VH Domain of                the monoclonal antibody capable of binding to the                epitope of a Disease Antigen (VH_(DA));            -   wherein the sub-Domains 1A and 1B are separated from one                another by a peptide Linker;        -   (ii) a Domain 2, wherein the Domain 2 is a            Heterodimer-Promoting Domain, wherein the            Heterodimer-Promoting Domain of the first and the second            polypeptide chains are different;    -   and wherein:    -   (a) the VL Domain of the first polypeptide chain and the VH        Domain of the second polypeptide chain associate to form the        Disease Antigen-Binding Domain, and the VH Domain of the first        polypeptide chain and the VL Domain of the second polypeptide        chain associate to form the CD3-Binding Domain; or    -   (b) the VL Domain of the first polypeptide chain and the VH        Domain of the second polypeptide chain associate to form the        CD3-Binding Domain, and the VH Domain of the first polypeptide        chain and the VL Domain of the second polypeptide chain        associate to form the Disease Antigen-Binding Domain.

The invention additionally concerns the embodiment of such DA×CD3Binding Molecule, wherein:

-   -   (a) said Heterodimer-Promoting Domain of said first polypeptide        chain is an E-coil Domain, and said Heterodimer-Promoting Domain        of said second polypeptide chain is a K-coil Domain; or    -   (b) said Heterodimer-Promoting Domain of said first polypeptide        chain is a K-coil Domain, and said Heterodimer-Promoting Domain        of said second polypeptide chain is an E-coil Domain.

The invention additionally concerns the embodiment of such DA×CD3Binding Molecule, wherein the first or second polypeptide chainadditionally comprises a Domain 3 comprising a CH2 and CH3 Domain of animmunoglobulin Fc Domain.

The invention additionally concerns the embodiment of such DA×CD3Binding Molecule, wherein the DA×CD3 Binding Molecule further comprisesa third polypeptide chain comprising a CH2 and CH3 Domain of animmunoglobulin Fc Domain.

The invention additionally concerns the embodiment of such DA×CD3Binding Molecule, wherein the DA×CD3 Binding Molecule further comprisesa CD8-Binding Domain.

The invention additionally concerns the embodiment of such DA×CD3Binding Molecule, wherein the DA×CD3 Binding Molecule comprises:

-   -   (I) (A) a first polypeptide comprising SEQ ID NO:179;        -   (B) a second polypeptide comprising SEQ ID NO:175; and        -   (C) a third polypeptide comprising SEQ ID NO:176; or    -   (II) (A) a first polypeptide comprising SEQ ID NO:184;        -   (B) a second polypeptide comprising SEQ ID NO:181; and        -   (C) a third polypeptide comprising SEQ ID NO:176; or    -   (III) (A) a first polypeptide comprising SEQ ID NO:196;        -   (B) a second polypeptide comprising SEQ ID NO:186; and        -   (C) a third polypeptide comprising SEQ ID NO:176; or    -   (IV) (A) a first polypeptide comprising SEQ ID NO:197;        -   (B) a second polypeptide comprising SEQ ID NO:192; and        -   (C) a third polypeptide comprising SEQ ID NO:176; or    -   (V) (A) a first polypeptide comprising SEQ ID NO:193;        -   (B) a second polypeptide comprising SEQ ID NO:194; and        -   (C) a third polypeptide comprising SEQ ID NO:176; or    -   (VI) (A) a first polypeptide comprising SEQ ID NO:179;        -   (B) a second polypeptide comprising SEQ ID NO:175;        -   (C) a third polypeptide comprising SEQ ID NO:187; and        -   (D) a fourth polypeptide comprising SEQ ID NO:188; or    -   (VII) (A) a first polypeptide comprising SEQ ID NO:184;        -   (B) a second polypeptide comprising SEQ ID NO:181;        -   (C) a third polypeptide comprising SEQ ID NO:187; and        -   (D) a fourth polypeptide comprising SEQ ID NO:188; or    -   (VIII) (A) a first polypeptide comprising SEQ ID NO:196;        -   (B) a second polypeptide comprising SEQ ID NO:186;        -   (C) a third polypeptide comprising SEQ ID NO:187; and        -   (D) a fourth polypeptide comprising SEQ ID NO:188; or    -   (IX) (A) a first polypeptide comprising SEQ ID NO:193;        -   (B) a second polypeptide comprising SEQ ID NO:194;        -   (C) a third polypeptide comprising SEQ ID NO:187; and        -   (D) a fourth polypeptide comprising SEQ ID NO:188.

The invention additionally concerns a pharmaceutical composition thatcomprises any of the above-described DA×CD3 Binding Molecules and apharmaceutically acceptable carrier.

The invention additionally concerns a method for the treatment of adisease, comprising administering to a subject in need thereof atherapeutically effective amount of any of the above-described DA×CD3Binding Molecules or the above-described pharmaceutical composition.

The invention additionally concerns the embodiment of such method,wherein the disease is cancer. Including embodiments, wherein the canceris selected from the group consisting of adrenal cancer, bladder cancer,breast cancer, colorectal cancer, gastric cancer, glioblastoma, kidneycancer, non-small-cell lung cancer, hematological cancer, multiplemyeloma, melanoma, ovarian cancer, pancreatic cancer, prostate cancer,skin cancer, renal cell carcinoma, testicular cancer, and uterinecancer.

The invention additionally concerns the embodiment of such method,wherein the disease is a pathogen-associated disease; includingembodiments, wherein the Pathogen-Associated Antigen is selected fromthe group consisting of the Pathogen-Associated Antigens: Herpes SimplexVirus infected cell protein (ICP)47, Herpes Simplex Virus gD,Epstein-Barr Virus LMP-1, Epstein-Barr Virus LMP-2A, Epstein-Barr VirusLMP-2B, Human Immunodeficiency Virus gp160, Human Immunodeficiency Virusgp120, Human Immunodeficiency Virus gp41, Human Papillomavirus E6, HumanPapillomavirus E7, human T-cell leukemia virus gp64, human T-cellleukemia virus gp46, and human T-cell leukemia virus gp21.

The invention additionally concerns the use of any of theabove-described DA×CD3 Binding Molecules or the above-describedpharmaceutical composition in the treatment of a disease.

The invention additionally concerns the embodiment of such use, whereinthe disease is cancer. Including, embodiments, wherein the cancer isselected from the group consisting of adrenal cancer, bladder cancer,breast cancer, colorectal cancer, gastric cancer, glioblastoma, kidneycancer, non-small-cell lung cancer, hematological cancer, multiplemyeloma, melanoma, ovarian cancer, pancreatic cancer, prostate cancer,skin cancer, renal cell carcinoma, testicular cancer, and uterinecancer.

The invention additionally concerns the embodiment of such use, whereinthe disease is a pathogen-associated disease. Including embodiments,wherein the Pathogen-Associated Antigen is selected from the groupconsisting of the Pathogen-Associated Antigens: Herpes Simplex Virusinfected cell protein (ICP)47, Herpes Simplex Virus gD, Epstein-BarrVirus LMP-1, Epstein-Barr Virus LMP-2A, Epstein-Barr Virus LMP-2B, HumanImmunodeficiency Virus gp160, Human Immunodeficiency Virus gp120, HumanImmunodeficiency Virus gp41, Human Papillomavirus E6, HumanPapillomavirus E7, human T-cell leukemia virus gp64, human T-cellleukemia virus gp46, and human T-cell leukemia virus gp21.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-1B provides schematics of representative covalently bondeddiabodies having two Epitope-Binding Domains composed of two polypeptidechains, each having an E-coil or K-coil Heterodimer-Promoting Domain(alternative Heterodimer-Promoting Domains are provided below). Acysteine residue may be present in a Linker (FIG. 1A) and/or in theHeterodimer-Promoting Domain (FIG. 1B). VL and VH Domains that recognizethe same epitope are shown using the same shading or fill pattern.

FIG. 2 provides a schematic of a representative covalently bondeddiabody molecule having two Epitope-Binding Domains composed of twopolypeptide chains, each having a CH2 and CH3 Domain, such that theassociated chains form all or part of an Fc Domain. VL and VH Domainsthat recognize the same epitope are shown using the same shading or fillpattern.

FIGS. 3A-3C provide schematics showing representative covalently bondedtetravalent diabodies having four Epitope-Binding Domains composed oftwo pairs of polypeptide chains (i.e., four polypeptide chains in all).One polypeptide of each pair possesses a CH2 and CH3 Domain, such thatthe associated chains form all or part of an Fc Domain. VL and VHDomains that recognize the same epitope are shown using the same shadingor fill pattern. The two pairs of polypeptide chains may be same. Insuch embodiments, wherein the two pairs of polypeptide chains are thesame and the VL and VH Domains recognize different epitopes (as shown inFIGS. 3A-3B), the resulting molecule possesses four Epitope-BindingDomains and is bispecific and bivalent with respect to each boundepitope. In such embodiments, wherein the VL and VH Domains recognizethe same epitope (e.g., the same VL Domain CDRs and the same VH DomainCDRs are used on both chains) the resulting molecule possesses fourEpitope-Binding Domains and is monospecific and tetravalent with respectto a single epitope. Alternatively, the two pairs of polypeptides may bedifferent. In such embodiments, wherein the two pairs of polypeptidechains are different and the VL and VH Domains of each pair ofpolypeptides recognize different epitopes (as shown by the differentshading and patterns in FIG. 3C), the resulting molecule possesses fourEpitope-Binding Domains and is tetraspecific and monovalent with respectto each bound epitope. FIG. 3A shows an Fc Domain-containing diabodywhich contains a peptide Heterodimer-Promoting Domain comprising acysteine residue. FIG. 3B shows an Fc Domain-containing diabody, whichcontains E-coil and K-coil Heterodimer-Promoting Domains comprising acysteine residue and a Linker (with an optional cysteine residue). FIG.3C, shows an Fc Domain-Containing diabody, which contains antibody CH1and CL Domains to promote heterdimerization.

FIGS. 4A-4B provide schematics of a representative covalently bondeddiabody molecule having two Epitope-Binding Domains composed of threepolypeptide chains. Two of the polypeptide chains possess a CH2 and CH3Domain, such that the associated chains form all or part of an FcDomain. The polypeptide chains comprising the VL and VH Domain furthercomprise a Heterodimer-Promoting Domain. VL and VH Domains thatrecognize the same epitope are shown using the same shading or fillpattern.

FIG. 5 provides the schematics of a representative covalently bondeddiabody molecule having four Epitope-Binding Domains composed of fivepolypeptide chains. Two of the polypeptide chains possess a CH2 and CH3Domain, such that the associated chains form an Fc Domain that comprisesall or part of an Fc Domain. The polypeptide chains comprising thelinked VL and VH Domains further comprise a Heterodimer-PromotingDomain. VL and VH Domains that recognize the same epitope are shownusing the same shading or fill pattern.

FIGS. 6A-6F provide schematics of representative Fc Domain-containingtrivalent Binding Molecules having three Epitope-Binding Domains. FIGS.6A and 6B, respectively, illustrate schematically the domains oftrivalent Binding Molecules comprising two Diabody-Type Binding Domainsand a Fab-Type Binding Domain having different domain orientations inwhich the Diabody-Type Binding Domains are N-terminal or C-terminal toan Fc Domain. The molecules in FIGS. 6A and 6B comprise four chains.FIGS. 6C and 6D, respectively, illustrate schematically the domains oftrivalent Binding Molecules comprising two Diabody-Type Binding DomainsN-terminal to an Fc Domain, and a Fab-Type Binding Domain in which theLight Chain and Heavy Chain are linked via a polypeptide spacer, or anscFv-Type Binding Domain. The trivalent Binding Molecules in FIGS. 6Eand 6F, respectively, illustrate schematically the domains of trivalentBinding Molecules comprising two Diabody-Type Binding Domains C-terminalto an Fc Domain, and a Fab-Type Binding Domain in which the Light Chainand Heavy Chain are linked via a polypeptide spacer, or an scFv-TypeBinding Domain. The trivalent Binding Molecules in FIGS. 6C-6F comprisethree chains. VL and VH Domains that recognize the same epitope areshown using the same shading or fill pattern.

FIG. 7A-7D show the results of CTL and binding assays. FIG. 7A shows theresults of representative redirected cell killing (% cytotoxicity in aCTL assay) mediated by DART-A-type diabody constructs containing the VLand VH Domains of CD3 mAb 1; CD3 mAb 1 M1; CD3 mAb 1 M2; CD3 mAb 1 M15;CD3 mAb 1 M17; CD3 mAb 1 M18; CD3 mAb 1 M19; and CD3 mAb 1 M20. FIGS.7B-7C plot the correlation between the affinity constants (FIG. 7B: KD;FIG. 7C: ka; and FIG. 7D: kd) and CTL activity (EC₅₀ of cytolysis at 18hours) reported in Tables 11-12.

FIG. 8A-8E show the results of representative studies of redirected cellkilling (CTL assay) mediated DART A-type diabody constructs containingthe VL and VH Domains of CD3 mAb 1; CD3 mAb 1 M2; CD3 mAb 1 M7; CD3 mAb1 M13; and CD3 mAb 1 M15; using Pan-T effector cells and MV-4-11leukemia target cells. Percent cytotoxicity is plotted in FIG. 8A.Cytokine responses are plotted in FIGS. 8B-8E (FIG. 8B: IFN-gamma; FIG.8C: TNF-alpha; FIG. 8D: IL-6; FIG. 8D: IL-2); NegCtrl: negative control.

FIG. 9A-9B show the ability of DART-B-type diabodies to bind to DiseaseAntigens. FIG. 9A shows the ability of CD123-WT, CD123-M1, CD123-M2 andCD123-M18 DART-B-type diabodies to bind to CD123-expressing MOLM-13cells. FIG. 9B shows the ability of 5T4-WT, 5T4-M1, 5T4-M2, and 5T4-M18DART-B-type diabodies to bind to 5T4-expressing A498 cells. Binding wasdetected using biotinylated antibody specific for the diabodies' E/Kcoils and streptavidin-phycoerythrin (PE).

FIGS. 10A-10B show the ability of CD123-WT, CD123-M1, CD123-M2 andCD123-M18 DART-B-type diabodies to bind to CD8+ T-cells (FIG. 10A) andCD4+ T-cells (FIG. 10B).

FIGS. 11A-11Q show the results of representative studies of redirectedcell killing (CTL assay) mediated by CD123×CD3 DART B-type diabodyconstructs (possessing Fc Domains): CD123-WT (FIGS. 11B, 11F, 11J and11N), CD123-M2 (FIGS. 11C, 11G, 11K and 11O), CD123-M18 (FIGS. 11D, 11H,11L and 11P), HIV-WT (FIGS. 11E, 11I, 11M and 11Q), using Pan-T effectorcells and MOLM-13 acute monocytic leukemia (AML) target cells. Percentcytotoxicity is plotted in FIG. 11A. Cytokine responses and percentcytotoxicity are plotted in FIGS. 11B-11Q (FIGS. 11B-11E: IFN-gamma;FIGS. 11F-11I: TNF-alpha; FIGS. 11J-11M: IL-6; FIGS. 11N-11Q: IL-2).

FIGS. 12A-12E show the results of representative studies of redirectedcell killing (CTL assay) mediated by CD123×CD3 DART B-type diabodyconstructs (possessing Fc Domains) using PBMC effector cells and MOLM-13AML target cells. Percent cytotoxicity is plotted in FIG. 12A (E:T=15:1,24 h). Cytokine responses are plotted in FIGS. 12B-12E (FIG. 12B:IFN-gamma; FIG. 12C: TNF-alpha; FIG. 12D: IL-6; FIG. 12E: IL-2).

FIGS. 13A-13Q show the results of representative studies of redirectedcell killing (CTL assay) mediated by 5T4×CD3 DART B-type diabodyconstructs (possessing Fc Domains) 5T4-WT (FIGS. 13B, 13F, 13J and 13N),5T4-M2 (FIGS. 13C, 13G, 13K and 13O), 5T4-M18 (FIGS. 13D, 13H, 13L and13P), HIV-WT (FIGS. 13E, 13I, 13M and 13Q), using Pan-T effector cellsand A498 renal cell carcinoma target cells (E:T=5:1, 24 h). Cytotoxicityis plotted in FIG. 13A. Cytokine responses and percent cytotoxicity areplotted in FIGS. 13B-13Q (FIGS. 13B-13E: IFN-gamma; FIGS. 13F-13I:TNF-alpha; FIGS. 13J-13M: IL-6; FIGS. 13N-13Q: IL-2).

FIGS. 14A-14J show the results of representative studies of redirectedcell killing (CTL assay) mediated by CD19×CD3 DART B-type diabodyconstructs (possessing Fc Domains) using PBMCs (FIGS. 14A-14E) or Pan-Teffector cells (FIGS. 14F-14J) (E:T=30:1 for PBMCs and 10:1 forPan-T-cells, 24-48 h). Percent cytotoxicity (48 hrs) is plotted in FIG.14A (PBMCs) and FIG. 14F (Pan-T-cells). Cytokine responses at 48 hoursusing PBMCs are plotted in FIGS. 14B-14E (PBMCs) and FIGS. 14G-14J (PanT-cells) (FIGS. 14B and 14G: IFN-gamma; FIGS. 14C and 14H: TNF-alpha;FIGS. 14D and 14I: IL-6; FIGS. 14E and 14J: IL-2).

FIGS. 15A-15E show the ability of representative CD123×CD3 DART-B-typediabodies to mediate T-cell activation. T-cell activation was measuredby evaluating the ability of the diabodies to affect expression of CD25and CD69. Percent cytotoxicity is plotted in FIG. 15A. Activation ofCD4⁺ T-cells as determined by measuring CD25 is plotted in FIG. 15B.Activation of CD4⁺ T-cells as determined by measuring CD69 is plotted inFIG. 15C. Activation of CD8⁺ T-cells as determined by measuring CD25 isplotted in FIG. 15D. Activation of CD8⁺ T-cells as determined bymeasuring CD69 is plotted in FIG. 15E.

FIGS. 16A-16E show the ability of representative 5T4×CD3 DART-B-typediabodies to mediate T-cell activation. T-cell activation was measuredby evaluating the ability of the diabodies to affect expression of CD25and CD69. Percent cytotoxicity is plotted in FIG. 16A. Activation ofCD4⁺ T-cells as determined by measuring CD25 is plotted in FIG. 16B.Activation of CD4⁺ T-cells as determined by measuring CD69 is plotted inFIG. 16C. Activation of CD8⁺ T-cells as determined by measuring CD25 isplotted in FIG. 16D. Activation of CD8⁺ T-cells as determined bymeasuring CD69 is plotted in FIG. 16E.

FIGS. 17A-17B show the results of in vivo studies on the ability ofexemplary CD123×CD3 DART B-type diabody constructs to mediate thereduction of tumors in vivo. CD123-WT (50 μg/kg) or CD123-M18 (at 5μg/kg or 50 μg/kg) were provided to mice that had received the KG1Acells, and tumor volume was assessed over 35 days. FIG. 17A: CD4; FIG.17B: CD8.

FIGS. 18A-18D show the results of in vivo studies on the ability ofCD123×CD3 DART B-type diabody constructs to mediate the reduction oftumors in vivo. CD123-WT, CD123-M2 or CD123-M18 (at 0.5, 5 50, or 500μg/kg) were provided to mice that had received the KG1A cells, and tumorvolume was assessed over 35 days. FIG. 18A: CD123-WT; FIG. 18B:CD123-M2; FIG. 18C: CD123-M18; FIG. 18D: CD123-WT and CD123-M18 50 μg/kgand 500 μg/kg treatment groups.

FIGS. 19A-19D show the results of in vivo studies on the ability ofCD123×CD3 DART B-type diabody constructs to mediate the reduction oftumors in vivo. CD123-WT, CD123-M2 or CD123-M18 (at 0.5, 5 50, or 500μg/kg) were provided to mice that had received the MV4-11 cells, andtumor volume was assessed over 35 days. FIG. 19A: CD123-WT; FIG. 19B:CD123-M18; FIG. 19C: CD123-M2; FIG. 19D: CD123-WT, CD123-M2 andCD123-M18 500 μg/kg treatment groups.

FIGS. 20A-20B show the results of in vivo studies on the ability of5T4×CD3 DART B-type diabody constructs to mediate the reduction oftumors in vivo. 5T4-WT (at 10, 50, 100, or 500 μg/kg), 5T4-M18 (at 10,50, 100, or 500 μg/kg) or 5T4-M2 (at 500 g/kg) were provided to micethat had received the SKOV3 cells, and tumor volume was assessed over 45days. FIG. 20A: 5T4-WT; FIG. 20B: 5T4-M18 and 5T4-M2.

FIGS. 21A-21D show the results of in vivo studies on the cytokinerelease profile induced by CD123×CD3 DART-B-type diabodies. Serumcytokine levels (pg/ml) were evaluated six hours after administration ofCD123-WT, CD123-M2 or CD123-M18 (at 50, or 500 μg/kg) to mice that hadreceived the KG1A cells. FIG. 21A: IFN-γ; FIG. 21B: TNF-α; FIG. 21C:IL-6; and FIG. 21D: IL-2.

FIGS. 22A-22C show the ability of CD123×CD3×CD8 TRIVALENT-typemolecules, T-CD123-WT, T-CD123-M1, T-CD123-M2 and T-CD123-M18, to bindto cell surface antigens. FIG. 22A: binding to CD123-expressing MOLM-13cells; FIG. 22B: binding to CD4⁺ T-cells; FIG. 22C: binding to CD8⁺T-cells.

FIGS. 23A-23G show the results of representative studies of redirectedcell killing (CTL assay) mediated by T-CD123-WT, T-CD123-M1, T-CD123-M2and T-CD123-M18 CD123×CD3×CD8 TRIVALENT-type molecules using differentT-cell populations. Percent cytotoxicity using CD3⁺ Pan-T-cells (FIG.23A); CD4⁺ T-cells (FIG. 23B) and CD8⁺ T-cells (FIG. 23C). Cytokineresponses using CD3⁺ Pan-T-cells are plotted in FIGS. 23D-23G. FIG. 23D:IFN-γ; FIG. 23E: TNF-α; FIG. 23F: IL-6; and FIG. 23G: IL-2.

FIGS. 24A-24J show the serum cytokine levels, Ki67 expression, andclinical pathology marker levels observed in cynomolgus monkeys treatedwith CD123-M18 (10 mg/kg and 20 mg/kg) or CD123-WT (0.003 mg/kg). FIG.24A: IFN-7; FIG. 24B: TNF-α; FIG. 24C: IL-6; FIG. 24D: IL-2; FIG. 24E:IL-15; FIG. 24F: Ki67 positive CD4⁺ T-cells; FIG. 24G: Ki67 positiveCD8⁺ T-cells; FIG. 24H: platelet; FIG. 24I: C-reactive protein; FIG.24J: blood urea nitrogen.

FIGS. 25A-25G show the results of a representative study of AML blastdepletion mediated by DART-A-WT, CD123-WT, CD123-M1 and CD123-M18 inperipheral blood samples from an AML patient. FIG. 25A: AML 34⁺ blastcell count as a percent of control; FIG. 25B: CD4⁺ cell expansion; FIG.25C: CD8⁺ cell expansion; FIGS. 25D-G: Cytokine release (FIG. 25D:IFN-7; FIG. 25E: TNF-α; FIG. 25F: IL-6; and FIG. 25G: IL-2).

FIGS. 26A-26E show the results of representative studies of redirectedcell killing (CTL assay) mediated by CD123×CD3 diabody constructsCD123-WT, CD123-M1, CD123-M13, CD123-M17, CD123-M18, and CD123-M19 usingPan-T effector cells and MOLM-13 AML target cells (E:T=15:1, 48-96 hr).Cytotoxicity as a function of % LDH released is plotted in FIG. 26A.Cytokine responses are plotted in FIGS. 26B-26E (FIG. 26B: IFN-gamma;FIG. 26C: TNF-alpha; FIG. 26D: IL-6; FIG. 26E: IL-2).

FIGS. 27A-27D present the cumulative results from 4-7 redirected cellkilling assays (CTL assay) and cytokine release studies mediated byCD123×CD3 diabody constructs CD123-WT, CD123-M1, CD123-M13, CD123-M17,CD123-M18, CD123-M19, and DART-A-WT using Pan-T effector cells andMOLM-13 AML target cells (E:T=15:1, 48-96 hr). CTL activity EC₅₀ valuesin pM are plotted in FIG. 27A. CTL activity as a multiple of the EC₅₀value of CD123-WT is plotted in FIG. 27B. CTL activity Emax as a percentof CD123-WT) is plotted in FIG. 27C. The calculated Therapeutic Index(TI=E_(max) (CTL): E_(max) (cytokine)) normalized to CD123-WT is plottedin FIG. 27D.

FIGS. 28A-28B show the results of in vivo studies on the ability ofCD123×CD3 diabody constructs to mediate the reduction of tumors in vivo.CD123-WT (0.5 mg/kg), CD123-M18 or CD123-M13 (at 0.005, 0.05, 0.5 and 1mg/kg) were provided to mice that had received KG1A cells, and tumorvolume was assessed over 42 days. FIG. 28A: CD123-WT and CD123-M18. FIG.28B: CD123-WT and CD123-M13.

FIGS. 29A-29B show the results of in vivo studies on the ability ofCD123×CD3 diabody constructs to mediate the reduction of tumors in vivo.CD123-WT (0.05 mg/kg), CD123-M18 or CD123-M17 (at 0.005, 0.05, 0.5 and 1mg/kg) were provided to mice that had received KG1A cells, and tumorvolume was assessed over 42 days. FIG. 29A: CD123-WT and CD123-M18. FIG.29B: CD123-WT and CD123-M17.

FIGS. 30A-30B show the results of in vivo studies on the interleukin-2cytokine release profile induced by CD123×CD3 DART-B-type diabodies.Serum cytokine levels (pg/ml) were evaluated six hours afteradministration of CD123-WT (0.5 mg/kg), CD123-M13, CD123-M17 orCD123-M18 (at 0.05, 0.5 and 1 mg/kg) to mice that had received the KG1Acells. FIG. 30A: CD123-WT, CD123-M13, and CD123-M18; and FIG. 30B:CD123-WT, CD123-M17 and CD123-M18.

FIGS. 31A-31F show the results of a representative study of autologousB-cell depletion by CD19-WT, CD19.1-M18, and HIV-M18 from human andcynomolgus monkey PBMCs. Depletion of CD20⁺ B-cells is plotted in FIG.31A (human PBMCs) and FIG. 31B (cyno PBMCs). Cytokine release from thetreated human PBMCs is plotted in FIGS. 31C-F (FIG. 31C: IFN-γ; FIG.31D: TNF-α; FIG. 31E: IL-6; and FIG. 31F: IL-2).

FIGS. 32A-32D show the reduction in B-cells levels observed in theperipheral blood of cynomolgus monkeys treated with CD19.1-M18 (1 mg/kgand 10 mg/kg) or CD123-WT (0.1 mg/kg). The predose B-cell levels areshow in FIG. 32A (the B-cell population is indicated with an oval). Thelevels at Day 1, Day 8 and Day 15 are shown in FIGS. 32B-32C,respectively.

FIGS. 33A-33C show the immunohistochemistry staining of B-cells in lymphnodes from cynomolgus monkeys pretreatment and at Day 7 post treatmentwith the positive control CD19-WT (FIG. 33A: 0.1 mg/kg) or the CD3variant CD19.1-M18 (FIG. 33B: 10 mg/kg; and FIG. 33C: 30 mg/kg).

FIG. 34 shows the reduction in B-cells levels observed in the peripheralblood of cynomolgus monkeys treated with CD19.1-M13 (1 mg/kg),CD19.1-M17 (1 mg/kg) or CD19-WT (0.1 mg/kg).

FIGS. 35A-35E show the serum cytokine levels observed in cynomolgusmonkeys treated with CD19.1-M13 (1 mg/kg), CD19.1-M17 (1 mg/kg), orCD19-WT (0.1 mg/kg). FIG. 35A: TNF-α, FIG. 35B: IFN-7, FIG. 35C: IL-2,FIG. 35D: IL-6; and FIG. 35E: IL-15.

FIGS. 36A-36B show the proliferation of T-cells observed in cynomolgusmonkeys treated with CD19.1-M13 (1 mg/kg), CD19.1-M17 (1 mg/kg), orCD19-WT (0.1 mg/kg). FIG. 36A: Ki67 positive T-cells CD4⁺ T-cells; FIG.36B: Ki67 positive CD8+ T-cells.

DETAILED DESCRIPTION OF THE INVENTION

The present invention is directed to multispecific Binding Molecules(e.g., a bispecific antibody, a diabody, a bispecific scFv, a trivalentmolecule, a TandAb®, a BiTE® etc.) comprising a CD3-Binding Domaincapable of binding an epitope of CD3 and also a Disease Antigen-BindingDomain capable of binding an epitope of a Disease Antigen (“DA”) (e.g.,a “DA×CD3 Binding Molecule”). The invention particularly concerns suchDA×CD3 Binding Molecules comprising a variant CD3-Binding Domain(“vCD3-Binding Domain”), which comprises a CDR_(H)1 Domain, a CDR_(H)2Domain, a CDR_(H)3 Domain, a CDR_(L)1 Domain, a CDR_(L)2 Domain, and aCDR_(L)3 Domain, at least one of which differs in amino acid sequencefrom the amino acid sequence of the corresponding CDR of a referenceCD3-Binding Domain (“rCD3-Binding Domain”), and wherein the DA×CD3Binding Molecule comprising such vCD3-Binding Domain exhibits an alteredaffinity for CD3, relative to a DA×CD3 Binding Molecule comprising suchrCD3-Binding Domain. The invention particularly concerns to such DA×CD3Binding Molecules comprising a vCD3-Binding Domain which exhibit reducedaffinity for CD3 and are capable of mediating redirected killing oftarget cells expressing a DA and exhibit lower levels of cytokinerelease relative to a DA×CD3 Binding Molecule comprising a rCD3-BindingDomain. The invention particularly concerns the use of DA×CD3 BindingMolecules comprising a vCD3-Binding Domain in the treatment of cancerand pathogen-associated diseases. The present invention is also directedto pharmaceutical compositions that comprise such molecule(s).

As indicated above, the therapeutic molecules of the present inventionparticularly include bispecific Binding Molecules that comprises anEpitope-Binding Domain capable of immunospecifically binding an epitopeof a cell surface molecule of an effector cell and an Epitope-BindingDomain that is capable of immunospecifically binding an epitope of atarget cell that expresses a Disease Antigen. As used herein, the term“Disease Antigen” (abbreviated as “DA”) denotes an antigen that isexpressed on the surface of an abnormal or infected cell and that ischaracteristic of such abnormality of infection, or that is expressed onthe surface of a foreign cell and that is characteristic of such foreignorigin. As used herein, a cell that expresses a Disease Antigen on itscell surface, and that may therefore become bound by the therapeuticmolecules of the present invention and thereby targeted for killing bysuch therapeutic molecules is a “target cell.” Of particular relevanceto the present invention are Disease Antigens that are “Cancer Antigens”or “Pathogen-Associated Antigens.”

I. Antibodies and their Binding Domains

The DA×CD3 Binding Molecules of the present invention may be antibodiesor be derivable from antibodies (e.g., by fragmentation, cleavage, etc.of antibody polypeptides, or from use of the amino acid sequences ofantibody molecules or of polynucleotides (or their sequences) thatencode such polynucleotides, etc.).

Antibodies are immunoglobulin molecules capable of specific binding to aparticular domain or moiety or conformation (an “epitope”) of amolecule, such as a carbohydrate, polynucleotide, lipid, polypeptide,etc. An epitope-containing molecule may have immunogenic activity, suchthat it elicits an antibody production response in an animal; suchmolecules are termed “antigens.” As used herein, the terms “antibody”and “antibodies” refer to monoclonal antibodies, multispecificantibodies, human antibodies, humanized antibodies, syntheticantibodies, chimeric antibodies, polyclonal antibodies, camelizedantibodies, single-chain Fvs (scFv), single-chain antibodies, Fabfragments, F(ab′) fragments, disulfide-linked bispecific Fvs (sdFv),intrabodies, and Epitope-Binding Domains of any of the above. Inparticular, the term “antibody” includes immunoglobulin molecules andimmunologically active fragments of immunoglobulin molecules, i.e.,molecules that contain an Epitope-Binding Domain. Immunoglobulinmolecules can be of any type (e.g., IgG, IgE, IgM, IgD, IgA and IgY),class (e.g., IgG₁, IgG₂, IgG₃, IgG₄, IgA₁ and IgA₂) or subclass.Antibodies are capable of “immunospecifically binding” to a polypeptideor protein or a non-protein molecule due to the presence on suchmolecule of a particular domain or moiety or conformation (an“epitope”).

The term “monoclonal antibody” refers to a homogeneous antibodypopulation wherein the monoclonal antibody is comprised of amino acids(naturally occurring or non-naturally occurring) that are involved inthe selective binding of an antigen. Monoclonal antibodies are highlyspecific, being directed against a single epitope (or antigenic site).The term “monoclonal antibody” encompasses not only intact monoclonalantibodies and full-length monoclonal antibodies, but also fragmentsthereof (such as Fab, Fab′, F(ab′)₂, Fv, single-chain (scFv), mutantsthereof), fusion proteins comprising an antibody portion, humanizedmonoclonal antibodies, chimeric monoclonal antibodies, and any othermodified configuration of the immunoglobulin molecule that comprises anantigen recognition site of the required specificity and the ability tobind to an antigen. It is not intended to be limited as regards to thesource of the antibody or the manner in which it is made (e.g., byhybridoma, phage selection, recombinant expression, transgenic animals,etc.). The term includes whole immunoglobulins as well as the fragmentsetc. described above under the definition of “antibody.” Methods ofmaking monoclonal antibodies are known in the art. One method which maybe employed is the method of Kohler, G. et al. (1975) “ContinuousCultures Of Fused Cells Secreting Antibody Of Predefined Specificity,”Nature 256:495-497 or a modification thereof. Typically, monoclonalantibodies are developed in mice, rats or rabbits. The antibodies areproduced by immunizing an animal with an immunogenic amount of cells,cell extracts, or protein preparations that contain the desired epitope.The immunogen can be, but is not limited to, primary cells, culturedcell lines, cancerous cells, proteins, peptides, nucleic acids, ortissue. Cells used for immunization may be cultured for a period of time(e.g., at least 24 hours) prior to their use as an immunogen. Cells maybe used as immunogens by themselves or in combination with anon-denaturing adjuvant, such as Ribi (see, e.g., Jennings, V. M. (1995)“Review of Selected Adjuvants Used in Antibody Production,” ILAR J.37(3):119-125). In general, cells should be kept intact and preferablyviable when used as immunogens. Intact cells may allow antigens to bebetter detected than ruptured cells by the immunized animal. Use ofdenaturing or harsh adjuvants, e.g., Freund's adjuvant, may rupturecells and therefore is discouraged. The immunogen may be administeredmultiple times at periodic intervals such as, bi weekly, or weekly, ormay be administered in such a way as to maintain viability in the animal(e.g., in a tissue recombinant). Alternatively, existing monoclonalantibodies and any other equivalent antibodies that are immunospecificfor a desired pathogenic epitope can be sequenced and producedrecombinantly by any means known in the art. In one embodiment, such anantibody is sequenced, and the polynucleotide sequence is then clonedinto a vector for expression or propagation. The sequence encoding theantibody of interest may be maintained in a vector in a host cell andthe host cell can then be expanded and frozen for future use. Thepolynucleotide sequence of such antibodies may be used for geneticmanipulation to generate the monospecific or multispecific (e.g.,bispecific, trispecific and tetraspecific) molecules of the invention aswell as an affinity optimized, a chimeric antibody, a humanizedantibody, and/or a caninized antibody, to improve the affinity, or othercharacteristics of the antibody, as detailed below.

The Binding Molecules of the present invention bind epitopes via theirbinding domains in an “immunospecific” manner. As used herein, anantibody, diabody or other epitope-binding molecule is said to“immunospecifically” bind a region of another molecule (i.e., anepitope) if it reacts or associates more frequently, more rapidly, withgreater duration and/or with greater affinity with that epitope relativeto alternative epitopes. For example, an antibody thatimmunospecifically binds to a viral epitope is an antibody that bindsthis viral epitope with greater affinity, avidity, more readily, and/orwith greater duration than it immunospecifically binds to other viralepitopes or non-viral epitopes. It is also understood by reading thisdefinition that, for example, an antibody (or moiety or epitope) thatimmunospecifically binds to a first target may or may not specificallyor preferentially bind a second target. As such, “immunospecificbinding” does not necessarily require (although it can include)exclusive binding. Generally, but not necessarily, reference herein tobinding means “immunospecific” binding.

The last few decades have seen a revival of interest in the therapeuticpotential of antibodies, and antibodies have become one of the leadingclasses of biotechnology-derived drugs (Chan, C. E. et al. (2009) “TheUse Of Antibodies In The Treatment Of Infectious Diseases,” SingaporeMed. J. 50(7):663-666). Over 200 antibody-based drugs have been approvedfor use or are under development.

Natural antibodies (such as IgG antibodies) are composed of two “LightChains” complexed with two “Heavy Chains.” Each Light Chain contains aVariable Domain (“VL”) and a Constant Domain (“CL”). Each Heavy Chaincontains a Variable Domain (“VH”), three Constant Domains (“CH1,” “CH2”and “CH3”), and a “Hinge” Region (“H”) located between the CH1 and CH2Domains. In contrast, scFvs are single-chain molecules made by linkingLight and Heavy Chain Variable Domains together via a short linkingpeptide.

The basic structural unit of naturally occurring immunoglobulins (e.g.,IgG) is thus a tetramer having two Light Chains and two Heavy Chains,usually expressed as a glycoprotein of about 150,000 Da. Theamino-terminal (“N-terminal”) portion of each chain includes a VariableDomain of about 100 to 110 or more amino acids primarily responsible forantigen recognition. The carboxy-terminal (“C-terminal”) portion of eachchain defines a constant region, with Light Chains having a singleConstant Domain and Heavy Chains usually having three Constant Domainsand a Hinge Domain. Thus, the structure of the Light Chains of an IgGmolecule is n-VL-CL-c and the structure of the IgG Heavy Chains isn-VH-CH1-H-CH2-CH3-c (where n and c represent, respectively, theN-terminus and the C-terminus of the polypeptide). The ability of anintact, unmodified antibody (e.g., an IgG antibody) to bind an epitopeof an antigen depends upon the presence and sequences of the VariableDomains. Unless specifically noted, the order of domains of the proteinmolecules described herein is in the “N-terminal to C-terminal”direction.

A. Characteristics of Antibody Variable Domains

The Variable Domains of an IgG molecule consist of three complementaritydetermining regions (“CDR”), which contain the amino acid residues ofthe antibody that will be in contact with epitope, and four interveningnon-CDR segments, referred to as framework regions (“FR”), whichseparate the CDR segments and which in general maintain the structureand determine the positioning of the CDR residues so as to permit themto contact the epitope (although certain framework residues may alsoplay a role in such contact). Thus, the VL and VH Domains have thestructure n-FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4-c, where n and c respectivelydenote the N-terminal end and the C-terminal end of the domains. Theamino acid sequences of the CDRs determine whether an antibody will beable to bind to a particular epitope.

Amino acids from the Variable Domains of the mature Heavy and LightChains of immunoglobulins are designated by the position of an aminoacid in the chain. Kabat et al. (SEQUENCES OF PROTEINS OF IMMUNOLOGICALINTEREST, 5^(th) Ed. Public Health Service, NH1, MD (1991) (“Kabat”),expressly incorporated herein by reference), described numerous aminoacid sequences for antibodies, identified an amino acid consensussequence for each subgroup, and assigned a residue number to each aminoacid. The CDRs are identified as defined by Kabat (it will be understoodthat CDR_(H)1 as defined by Chothia, C. & Lesk, A. M. ((1987) “Canonicalstructures for the hypervariable regions of immunoglobulins,” J. Mol.Biol. 196:901-917) begins five residues earlier). Kabat's numberingscheme is extendible to antibodies not included in his compendium byaligning the antibody in question with one of the consensus sequences inKabat by reference to conserved amino acids. This method for assigningresidue numbers has become standard in the field and readily identifiesamino acids at equivalent positions in different antibodies, includingchimeric or humanized variants. For example, an amino acid at position50 of a human antibody Light Chain occupies the equivalent position toan amino acid at position 50 of a mouse antibody Light Chain.

Polypeptides that are (or may serve as) the first, second and third CDRof the Light Chain of an antibody are herein respectively designated as:CDR_(L)1 Domain, CDR_(L)2 Domain, and CDR_(L)3 Domain. Similarly,polypeptides that are (or may serve as) the first, second and third CDRof the Heavy Chain of an antibody are herein respectively designated as:CDR_(H)1 Domain, CDR_(H)2 Domain, and CDR_(H)3 Domain. Thus, the termsCDR_(L)1 Domain, CDR_(L)2 Domain, CDR_(L)3 Domain, CDR_(H)1 Domain,CDR_(H)2 Domain, and CDR_(H)3 Domain are directed to polypeptides thatwhen incorporated into a protein cause that protein to be able to bind aspecific epitope regardless of whether such protein is an antibodyhaving light and Heavy Chains or is a diabody or a single-chain bindingmolecule (e.g., an scFv, a BiTe, etc.), or is another type of protein.Accordingly, as used herein, the term “Epitope-Binding Domain” denotes adomain comprising a fragment or portion of a binding molecule (or apolypeptide having the amino acid sequence of such a fragment orportion) that contributes to the ability of the binding molecule toimmunospecifically bind an epitope. An Epitope-Binding Domain maycontain any 1, 2, 3, 4, or 5 the CDR Domains of an antibody, or maycontain all 6 of the CDR Domains of an antibody and, although capable ofimmunospecifically binding such epitope, may exhibit animmunospecificity, affinity or selectivity towards such epitope thatdiffers from that of such antibody. An Epitope-Binding Domain maycontain only part of a CDR, namely the subset of CDR residues requiredfor binding (termed “Specificity-Determining Residues,” or “SDRs;” Kim,J. H. et al. (2012) “Humanization By CDR Grafting AndSpecificity-Determining Residue Grafting,” Methods Mol. Biol.907:237-245; Kim, K. S. et al. (2010) “Construction Of A HumanizedAntibody To Hepatitis B Surface Antigen By Specificity-DeterminingResidues (SDR)-Grafting And De-Immunization,” Biochem. Biophys. Res.Commun. 396(2):231-237; Kashmiri, S. V. et al. (2005) “SDR Grafting—ANew Approach To Antibody Humanization,” Methods 36(1):25-34; Gonzales,N. R. et al. (2004) “SDR Grafting Of A Murine Antibody Using MultipleHuman Germline Templates To Minimize Its Immunogenicity,” Mol. Immunol.41:863-872). Preferably, however, an Epitope-Binding Domain will containall 6 of the CDR Domains of such antibody. An Epitope-Binding Domain ofan antibody may be a single polypeptide chain (e.g., an scFv), or maycomprise two or more polypeptide chains, each having an amino terminusand a carboxy terminus (e.g., a diabody, a Fab fragment, an Fab₂fragment, etc.).

The invention also particularly encompasses Binding Molecules thatcomprise a VL and/or VH Domain of a humanized antibody. The term“humanized antibody” refers to a chimeric molecule, generally preparedusing recombinant techniques, having an Epitope-Binding Domain of animmunoglobulin from a non-human species and a remaining immunoglobulinstructure of the molecule that is based upon the structure and/orsequence of a human immunoglobulin. The polynucleotide sequence of theVariable Domains of such antibodies may be used for genetic manipulationto generate such derivatives and to improve the affinity, or othercharacteristics of such antibodies. The general principle in humanizingan antibody involves retaining the basic sequence of the Epitope-BindingDomain of the antibody, while swapping the non-human remainder of theantibody with human antibody sequences. There are four general steps tohumanize a monoclonal antibody. These are: (1) determining thenucleotide and predicted amino acid sequence of the starting antibodylight and heavy Variable Domains (2) designing the humanized antibody orcaninized antibody, i.e., deciding which antibody framework region touse during the humanizing or canonizing process (3) the actualhumanizing or caninizing methodologies/techniques and (4) thetransfection and expression of the humanized antibody. See, for example,U.S. Pat. Nos. 4,816,567; 5,807,715; 5,866,692; and 6,331,415

The Epitope-Binding Domain may comprise either a complete VariableDomain fused onto Constant Domains or only the complementaritydetermining regions (CDRs) of such Variable Domain grafted toappropriate framework regions. Epitope-binding domains may be wild-typeor modified by one or more amino acid substitutions. This eliminates theconstant region as an immunogen in human individuals, but thepossibility of an immune response to the foreign Variable Domain remains(LoBuglio, A. F. et al. (1989) “Mouse/Human Chimeric Monoclonal AntibodyIn Man: Kinetics And Immune Response,” Proc. Natl. Acad. Sci. (U.S.A.)86:4220-4224). Another approach focuses not only on providinghuman-derived constant regions, but modifying the Variable Domains aswell so as to reshape them as closely as possible to human form. It isknown that the Variable Domains of both Heavy and Light Chains containthree complementarity determining regions (CDRs) which vary in responseto the antigens in question and determine binding capability, flanked byfour framework regions (FRs) which are relatively conserved in a givenspecies and which putatively provide a scaffolding for the CDRs. Whennon-human antibodies are prepared with respect to a particular antigen,the Variable Domains can be “reshaped” or “humanized” by grafting CDRsderived from non-human antibody on the FRs present in the human antibodyto be modified. Application of this approach to various antibodies hasbeen reported by Sato, K. et al. (1993) Cancer Res 53:851-856.Riechmann, L. et al. (1988) “Reshaping Human Antibodies for Therapy,”Nature 332:323-327; Verhoeyen, M. et al. (1988) “Reshaping HumanAntibodies: Grafting An Antilysozyme Activity,” Science 239:1534-1536;Kettleborough, C. A. et al. (1991) “Humanization Of A Mouse MonoclonalAntibody By CDR-Grafting: The Importance Of Framework Residues On LoopConformation,” Protein Engineering 4:773-3783; Maeda, H. et al. (1991)“Construction Of Reshaped Human Antibodies With HIV-NeutralizingActivity,” Human Antibodies Hybridoma 2:124-134; Gorman, S. D. et al.(1991) “Reshaping A Therapeutic CD4 Antibody,” Proc. Natl. Acad. Sci.(U.S.A.) 88:4181-4185; Tempest, P. R. et al. (1991) “Reshaping A HumanMonoclonal Antibody To Inhibit Human Respiratory Syncytial VirusInfection in vivo,” Bio/Technology 9:266-271; Co, M. S. et al. (1991)“Humanized Antibodies For Antiviral Therapy,” Proc. Natl. Acad. Sci.(U.S.A.) 88:2869-2873; Carter, P. et al. (1992) “Humanization Of AnAnti-p185her2 Antibody For Human Cancer Therapy,” Proc. Natl. Acad. Sci.(U.S.A.) 89:4285-4289; and Co, M. S. et al. (1992) “Chimeric AndHumanized Antibodies With Specificity For The CD33 Antigen,” J. Immunol.148:1149-1154. In some embodiments, humanized antibodies preserve allCDR sequences (for example, a humanized mouse antibody which containsall six CDRs from the mouse antibodies). In other embodiments, humanizedantibodies have one or more CDRs (one, two, three, four, five, or six)which differ in sequence relative to the original antibody.

A number of humanized antibody molecules comprising an Epitope-BindingDomain derived from a non-human immunoglobulin have been described,including chimeric antibodies having rodent or modified rodent VariableDomain and their associated complementarity determining regions (CDRs)fused to human Constant Domains (see, for example, Winter et al. (1991)“Man-made Antibodies,” Nature 349:293-299; Lobuglio et al. (1989)“Mouse/Human Chimeric Monoclonal Antibody In Man: Kinetics And ImmuneResponse,” Proc. Natl. Acad. Sci. (U.S.A.) 86:4220-4224 (1989), Shaw etal. (1987) “Characterization Of A Mouse/Human Chimeric MonoclonalAntibody (17-1A) To A Colon Cancer Tumor-Associated Antigen,” J.Immunol. 138:4534-4538, and Brown et al. (1987) “Tumor-SpecificGenetically Engineered Murine/Human Chimeric Monoclonal Antibody,”Cancer Res. 47:3577-3583). Other references describe rodent CDRs graftedinto a human supporting framework region (FR) prior to fusion with anappropriate human antibody Constant Domain (see, for example, Riechmann,L. et al. (1988) “Reshaping Human Antibodies for Therapy,” Nature332:323-327; Verhoeyen, M. et al. (1988) “Reshaping Human Antibodies:Grafting An Antilysozyme Activity,” Science 239:1534-1536; and Jones etal. (1986) “Replacing The Complementarity-Determining Regions In A HumanAntibody With Those From A Mouse,” Nature 321:522-525). Anotherreference describes rodent CDRs supported by recombinantly veneeredrodent framework regions. See, for example, European Patent PublicationNo. 519,596. These “humanized” molecules are designed to minimizeunwanted immunological response towards rodent anti-human antibodymolecules, which limits the duration and effectiveness of therapeuticapplications of those moieties in human recipients. Other methods ofhumanizing antibodies that may also be utilized are disclosed byDaugherty et al. (1991) “Polymerase Chain Reaction Facilitates TheCloning, CDR-Grafting, And Rapid Expression Of A Murine MonoclonalAntibody Directed Against The CD18 Component Of Leukocyte Integrins,”Nucl. Acids Res. 19:2471-2476 and in U.S. Pat. Nos. 6,180,377;6,054,297; 5,997,867; and 5,866,692.

B. Characteristics of Antibody Constant Regions

Throughout the present specification, the numbering of the residues inthe constant region of an IgG is that of the EU index as in Kabat etal., SEQUENCES OF PROTEINS OF IMMUNOLOGICAL INTEREST, 5^(th) Ed. PublicHealth Service, NH1, MD (1991) (“Kabat”), expressly incorporated hereinby reference. The term “EU index as in Kabat” refers to the numbering ofthe Constant Domains of human IgG1 EU antibody.

Polymorphisms have been observed at a number of different positionswithin antibody constant regions (e.g., Fc positions, including but notlimited to positions 270, 272, 312, 315, 356, and 358 as numbered by theEU index as set forth in Kabat), and thus slight differences between thepresented sequence and sequences in the prior art can exist. Polymorphicforms of human immunoglobulins have been well-characterized. At present,18 Heavy Chain allotypes (“Gm allotypes”) are known: G1m (1, 2, 3, 17)or G1m (a, x, f, z), G2m (23) or G2m (n), G3m (5, 6, 10, 11, 13, 14, 15,16, 21, 24, 26, 27, 28) or G3m (b1, c3, b3, b0, b3, b4, s, t, g1, c5, u,v, g5) (Lefranc, et al., “The Human IgG Subclasses: Molecular AnalysisOf Structure, Function And Regulation.” Pergamon, Oxford, pp. 43-78(1990); Lefranc, G. et al., 1979, Hum. Genet.: 50, 199-211). It isspecifically contemplated that the antibodies of the present inventionmay incorporate any allotype, isoallotype, or haplotype of anyimmunoglobulin gene, and are not limited to the allotype, isoallotype orhaplotype of the sequences provided herein. Furthermore, in someexpression systems the C-terminal amino acid residue (bolded above) ofthe CH3 Domain may be post-translationally removed. Accordingly, theC-terminal residue of the CH3 Domain is an optional amino acid residuein the Binding Molecules of the invention. Specifically encompassed bythe instant invention are Binding Molecules lacking the C-terminalresidue of the CH3 Domain. Also specifically encompassed by the instantinvention are such constructs comprising the C-terminal lysine residueof the CH3 Domain.

1. Constant Regions of the Heavy Chain

The CH1 Domains of the two Heavy Chains of an antibody complex with theantibody's Light Chain's “CL” constant region, and are attached to theHeavy Chains CH2 Domains via an intervening Hinge Domain.

An exemplary CH1 Domain is a human IgG1 CH1 Domain. The amino acidsequence of an exemplary human IgG1 CH1 Domain is (SEQ ID NO:1):

ASTKGPSVFP LAPSSKSTSG GTAALGCLVK DYFPEPVTVS WNSGALTSGV HTFPAVLQSS GLYSLSSVVT VPSSSLGTQT  YICNVNHKPS NTKVDKRV 

An exemplary CH1 Domain is a human IgG2 CH1 Domain. The amino acidsequence of an exemplary human IgG2 CH1 Domain is (SEQ ID NO:2):

ASTKGPSVFP LAPCSRSTSE STAALGCLVK DYFPEPVTVS WNSGALTSGV HTFPAVLQSS GLYSLSSVVT VPSSNFGTQT  YTCNVDHKPS NTKVDKTV 

An exemplary CH1 Domain is a human IgG3 CH1 Domain. The amino acidsequence of an exemplary human IgG3 CH1 Domain is (SEQ ID NO:3):

ASTKGPSVFP LAPCSRSTSG GTAALGCLVK DYFPEPVTVS WNSGALTSGV HTFPAVLQSS GLYSLSSVVT VPSSSLGTQT  YTCNVNHKPS NTKVDKRV 

An exemplary CH1 Domain is a human IgG4 CH1 Domain. The amino acidsequence of an exemplary human IgG4 CH1 Domain is (SEQ ID NO:4):

ASTKGPSVFP LAPCSRSTSE STAALGCLVK DYFPEPVTVS WNSGALTSGV HTFPAVLQSS GLYSLSSVVT VPSSSLGTKT  YTCNVDHKPS NTKVDKRV 

One exemplary Hinge Domain is a human IgG1 Hinge Domain. The amino acidsequence of an exemplary human IgG1 Hinge Domain is (SEQ ID NO:5):

EPKSCDKTHTCPPCP. 

Another exemplary Hinge Domain is a human IgG2 Hinge Domain. The aminoacid sequence of an exemplary human IgG2 Hinge Domain is (SEQ ID NO:6):

ERKCCVECPPCP. 

Another exemplary Hinge Domain is a human IgG3 Hinge Domain. The aminoacid sequence of an exemplary human IgG2 Hinge Domain is (SEQ ID NO:7):

ELKTPLGDTT HTCPRCPEPK SCDTPPPCPR CPEPKSCDTP  PPCPRCPEPK SCDTPPPCPR CP 

Another exemplary Hinge Domain is a human IgG4 Hinge Domain. The aminoacid sequence of an exemplary human IgG4 Hinge Domain is (SEQ ID NO:8):ESKYGPPCPSCP. As described herein, an IgG4 Hinge Domain may comprise astabilizing mutation such as the S228P substitution. The amino acidsequence of an exemplary S228P-stabilized human IgG4 Hinge Domain is(SEQ ID NO:9): ESKYGPPCPPCP.

The CH2 and CH3 Domains of the two Heavy Chains of an IgG antibodyinteract to form an “Fc Domain,” of IgG antibodies that is recognized bycellular Fc Receptors, including but not limited to Fc gamma Receptors(FcγRs). As used herein, the term “Fc Domain” is used to define aC-terminal region of an IgG Heavy Chain. An Fc Domain is said to be of aparticular IgG isotype, class or subclass if its amino acid sequence ismost homologous to that isotype relative to other IgG isotypes. Inaddition to their known uses in diagnostics, antibodies have been shownto be useful as therapeutic agents.

The amino acid sequence of the CH2-CH3 Domain of an exemplary human IgG1is (SEQ ID NO:10):

231      240        250        260        270        280 APELLGGPSV FLFPPKPKDT LMISRTPEVT CVVVDVSHED PEVKFNWYVD          290        300        310        320        330 GVEVHNAKTK PREEQYNSTY RVVSVLTVLH QDWLNGKEYK CKVSNKALPA          340        350        360        370        380 PIEKTISKAK GQPREPQVYT LPPSREEMTK NQVSLTCLVK GFYPSDIAVE          390        400        410        420        430 WESNGQPENN YKTIPPVLDS DGSFFLYSKL TVDKSRWQQG NVFSCSVMHE          440     447  ALHNHYTQKS LSLSPG X  

-   -   as numbered by the EU index as set forth in Kabat, wherein X is        lysine (K) or is absent.

The amino acid sequence of the CH2-CH3 Domain of an exemplary human IgG2is (SEQ ID NO:11):

231      240        250        260        270        280 APPVA-GPSV FLFPPKPKDT LMISRTPEVT CVVVDVSHED PEVQFNWYVD          290        300        310        320        330 GVEVHNAKTK PREEQFNSTF RVVSVLTVVH QDWLNGKEYK CKVSNKGLPA          340        350        360        370        380 PIEKTISKTK GQPREPQVYT LPPSREEMTK NQVSLTCLVK GFYPSDISVE          390        400        410        420        430 WESNGQPENN YKTTPPMLDS DGSFFLYSKL TVDKSRWQQG NVFSCSVMHE          440     447  ALHNHYTQKS LSLSPG X  

-   -   as numbered by the EU index as set forth in Kabat, wherein X is        lysine (K) or is absent.

The amino acid sequence of the CH2-CH3 Domain of an exemplary human IgG3is (SEQ ID NO:12):

231      240        250        260        270        280 APFLLGGPSV FLFPPKPKDT LMISRTPEVT CVVVDVSHED PEVQFKWYVD          290        300        310        320        330 GVEVHNAKTK PREEQYNSTF RVVSVLTVLH QDWLNGKEYK CKVSNKALPA          340        350        360        370        380 PIEKTISKTK GQPREPQVYT LPPSREEMTK NQVSLTCLVK GFYPSDIAVE          390        400        410        420        430 WESSGQPENN YNTTPPMLDS DGSFFLYSKL TVDKSRWQQG NIFSCSVMHE          440     447  ALHNRFTQKS LSLSPG X  

-   -   as numbered by the EU index as set forth in Kabat, wherein X is        lysine (K) or is absent.

The amino acid sequence of the CH2-CH3 Domain of an exemplary human IgG4is (SEQ ID NO:13):

231      240        250        260        270        280 APEFLGGPSV FLFPPKPKDT LMISRTPEVT CVVVDVSQED PEVQFNWYVD          290        300        310        320        330 GVEVHNAKTK PREEQFNSTY RVVSVLTVLH QDWLNGKEYK CKVSNKGLPS          340        350        360        370        380 SIEKTISKAK GQPREPQVYT LPPSQEEMTK NQVSLTCLVK GFYPSDIAVE          390        400        410       420         430 WESNGQPENN YKTIPPVLDS DGSFFLYSRL TVDKSRWQEG NVFSCSVMHE          440     447  ALHNHYTQKS LSLSLG X  

-   -   as numbered by the EU index as set forth in Kabat, wherein X is        lysine (K) or is absent.

2. Constant Regions of the Light Chain

As indicated above, each Light Chain of an antibody contains a VariableDomain (“VL”) and a Constant Domain (“CL”).

A preferred CL Domain is a human IgG CL Kappa Domain. The amino acidsequence of an exemplary human CL Kappa Domain is (SEQ ID NO:14):

RTVAAPSVFI FPPSDEQLKS GTASVVCLLN NFYPREAKVQ WKVDNALQSG NSQESVIEQD SKDSTYSLSS TLTLSKADYE  KHKVYACEVT HQGLSSPVTK SFNRGEC 

Alternatively, an exemplary CL Domain is a human IgG CL Lambda Domain.The amino acid sequence of an exemplary human CL Lambda Domain is (SEQID NO:15):

QPKAAPSVTL FPPSSEELQA NKATLVCLIS DFYPGAVTVA WKADSSPVKA GVETTPSKQS NNKYAASSYL SLTPEQWKSH  RSYSCQVTHE GSTVEKTVAP TECS 

II. Multispecific Binding Molecules

The ability of an antibody to bind an epitope of an antigen depends uponthe presence and amino acid sequence of the antibody's VL and VHDomains. Interaction of an antibody's Light Chain and Heavy Chain and,in particular, interaction of its VL and VH Domains forms one of the twoEpitope-Binding Domains of a natural antibody, such as an IgG. Naturalantibodies are capable of binding only one epitope species (i.e., theyare monospecific), although they can bind multiple copies of thatspecies (i.e., exhibiting bivalency or multivalency).

The functionality of antibodies can be enhanced by generatingmultispecific antibody-based molecules that can simultaneously bind twoseparate and distinct antigens (or different epitopes of the sameantigen) and/or by generating antibody-based molecule having highervalency (i.e., more than two Epitope-Binding Domains) for the sameepitope and/or antigen.

In order to provide molecules having greater capability than naturalantibodies, a wide variety of recombinant bispecific antibody formatshave been developed (see, e.g., PCT Publication Nos. WO 2008/003116, WO2009/132876, WO 2008/003103, WO 2007/146968, WO 2009/018386, WO2012/009544, WO 2013/070565), most of which use Linker peptides eitherto fuse a further Epitope-Binding Domain (e.g., an scFv, VL, VH, etc.)to, or within the antibody core (IgA, IgD, IgE, IgG or IgM), or to fusemultiple Epitope-Binding Domains (e.g., two Fab fragments or scFvs).Alternative formats use Linker peptides to fuse Epitope-Binding Domains(e.g., an scFv, VL, VH, etc.) to a dimerization domain such as theCH2-CH3 Domain or alternative polypeptides (WO 2005/070966, WO2006/107786 WO 2006/107617, WO 2007/046893). PCT Publication Nos. WO2013/174873, WO 2011/133886 and WO 2010/136172 disclose a trispecificantibody in which the CL and CH1 Domains are switched from theirrespective natural positions and the VL and VH Domains have beendiversified (WO 2008/027236; WO 2010/108127) to allow them to bind morethan one antigen. PCT Publication Nos. WO 2013/163427 and WO 2013/119903disclose modifying the CH2 Domain to contain a fusion protein adductcomprising a binding domain. PCT Publication Nos. WO 2010/028797,WO2010028796 and WO 2010/028795 disclose recombinant antibodies whose FcDomains have been replaced with additional VL and VH Domains, so as toform trivalent Binding Molecules. PCT Publication Nos. WO 2003/025018and WO2003012069 disclose recombinant diabodies whose individual chainscontain scFv Domains. PCT Publication Nos. WO 2013/006544 disclosesmultivalent Fab molecules that are synthesized as a single polypeptidechain and then subjected to proteolysis to yield heterodimericstructures. PCT Publication Nos. WO 2014/022540, WO 2013/003652, WO2012/162583, WO 2012/156430, WO 2011/086091, WO 2008/024188, WO2007/024715, WO 2007/075270, WO 1998/002463, WO 1992/022583 and WO1991/003493 disclose adding additional binding domains or functionalgroups to an antibody or an antibody portion (e.g., adding a diabody tothe antibody's Light Chain, or adding additional VL and VH Domains tothe antibody's light and Heavy Chains, or adding a heterologous fusionprotein or chaining multiple Fab Domains to one another).

The art has additionally noted the capability to produce diabodies thatdiffer from such natural antibodies in being capable of binding two ormore different epitope species (i.e., exhibiting bispecificity ormultispecificity in addition to, or in exchange of, bivalency ormultivalency) (see, e.g., Holliger et al. (1993) “‘Diabodies’: SmallBivalent And Bispecific Antibody Fragments,” Proc. Natl. Acad. Sci.(U.S.A.) 90:6444-6448; US 2004/0058400 (Hollinger et al.); US2004/0220388/WO 02/02781 (Mertens et al.); Alt et al. (1999) FEBS Lett.454(1-2):90-94; Lu, D. et al. (2005) “A Fully Human Recombinant IgG-LikeBispecific Antibody To Both The Epidermal Growth Factor Receptor And TheInsulin-Like Growth Factor Receptor For Enhanced Antitumor Activity,” J.Biol. Chem. 280(20):19665-19672; WO 02/02781 (Mertens et al.); Olafsen,T. et al. (2004) “Covalent Disulfide-Linked Anti-CEA Diabody AllowsSite-Specific Conjugation And Radiolabeling For Tumor TargetingApplications,” Protein Eng. Des. Sel. 17(1):21-27; Wu, A. et al. (2001)“Multimerization Of A Chimeric Anti-CD20 Single Chain Fv-Fv FusionProtein Is Mediated Through Variable Domain Exchange,” ProteinEngineering 14(2):1025-1033; Asano et al. (2004) “A Diabody For CancerImmunotherapy And Its Functional Enhancement By Fusion Of Human FcDomain,” Abstract 3P-683, J. Biochem. 76(8):992; Takemura, S. et al.(2000) “Construction Of A Diabody (Small Recombinant BispecificAntibody) Using A Refolding System,” Protein Eng. 13(8):583-588;Baeuerle, P. A. et al. (2009) “Bispecific T-Cell Engaging Antibodies ForCancer Therapy,” Cancer Res. 69(12):4941-4944).

In particular, stable, covalently bonded heterodimeric non-monospecificdiabodies, termed DART® diabodies have been developed; see, e.g., Sloan,D. D. et al. (2015) “Targeting HIV Reservoir in Infected CD4 T Cells byDual-Affinity Re-targeting Molecules (DARTs) that Bind HIV Envelope andRecruit Cytotoxic T Cells,” PLoS Pathog. 11(11):e1005233. doi:10.1371/journal.ppat.1005233; Al Hussaini, M. et al. (2015) “TargetingCD123 In AML Using A T-Cell Directed Dual-Affinity Re-Targeting (DART®)Platform,” Blood pii: blood-2014-05-575704; Chichili, G. R. et al.(2015) “A CD3×CD123 Bispecific DART For Redirecting Host T Cells ToMyelogenous Leukemia: Preclinical Activity And Safety In NonhumanPrimates,” Sci. Transl. Med. 7(289):289ra82; Moore, P. A. et al. (2011)“Application Of Dual Affinity Retargeting Molecules To Achieve OptimalRedirected T-Cell Killing Of B-Cell Lymphoma,” Blood 117(17):4542-4551;Veri, M. C. et al. (2010) “Therapeutic Control Of B-Cell Activation ViaRecruitment Of Fcgamma Receptor IIb (CD32B) Inhibitory Function With ANovel Bispecific Antibody Scaffold,” Arthritis Rheum. 62(7):1933-1943;Johnson, S. et al. (2010) “Effector Cell Recruitment With Novel Fv-BasedDual-Affinity Re-Targeting Protein Leads To Potent Tumor Cytolysis Andin vivo B-Cell Depletion,” J. Mol. Biol. 399(3):436-449); U.S. Pat. Nos.8,044,180; 8,133,982; 8,187,593; 8,193,318; 8,530,627; 8,669,349;8,778,339; 8,784,808; 8,795,667; 8,802,091; 8,802,093; 8,946,387;8,968,730; and 8,993,730; US Patent Publication Nos. 2009/0060910;2010/0174053; 2011/0081347; 2011/0097323; 2011/0117089; 2012/0009186;2012/0034221; 2012/0141476; 2012/0294796; 2013/0149236; 2013/0295121;2014/0017237; and 2014/0099318; European Patent Documents No. EP1868650; EP 2158221; EP 2247304; EP 2252631; EP 2282770; EP 2328934; EP2376109; EP 2542256; EP 2601216; EP 2714079; EP 2714733; EP 2786762; EP2839842; EP 2840091; and PCT Publication Nos. WO 2006/113665; WO2008/157379; WO 2010/027797; WO 2010/033279; WO 2010/080538; WO2011/109400; WO 2012/018687; WO 2012/162067; WO 2012/162068; WO2014/159940; WO 2015/021089; WO 2015/026892; and WO 2015/026894). Suchdiabodies comprise two or more covalently complexed polypeptides andinvolve engineering one or more cysteine residues into each of theemployed polypeptide species that permit disulfide bonds to form andthereby covalently bond one or more pairs of such polypeptide chains toone another. For example, the addition of a cysteine residue to theC-terminus of such constructs has been shown to allow disulfide bondingbetween the involved polypeptide chains, stabilizing the resultingdiabody without interfering with the diabody's binding characteristics.

The simplest DART® diabody comprises two polypeptide chains eachcomprising three Domains (FIGS. 1A-1B). The first polypeptide chaincomprises: (i) a Domain that comprises an Epitope-Binding Domain of aLight Chain Variable Domain of the a first immunoglobulin (VL1), (ii) asecond Domain that comprises an Epitope-Binding Domain of a Heavy ChainVariable Domain of a second immunoglobulin (VH2), and (iii) a thirdDomain that serves to promote heterodimerization (a“Heterodimer-Promoting Domain”) with the second polypeptide chain and tocovalently bond the first polypeptide to the second polypeptide chain ofthe diabody. The second polypeptide chain contains a complementary firstDomain (a VL2 Domain), a complementary second Domain (a VH1 Domain) anda third Domain that complexes with the third Domain of the firstpolypeptide chain in order to promote heterodimerization (a“Heterodimer-Promoting Domain”) and covalent bonding with the firstpolypeptide chain. Such molecules are stable, potent and have theability to simultaneously bind two or more antigens. In one embodiment,the Third Domains of the first and second polypeptide chains eachcontain a cysteine residue (denoted as “©” in the Figures), which servesto bind the polypeptides together via a covalent disulfide bond. Thethird Domain of one or both of the polypeptide chains may additionallypossess the sequence of a CH2-CH3 Domain, such that complexing of onediabody polypeptide to another diabody polypeptide forms an Fc Domain.Such Fc Domains may serve to alter the biological half-life of thediabody, decrease its immunogenicity, and/or be capable of binding to anFc Receptor of cells (such as B lymphocytes, dendritic cells, naturalkiller cells, macrophages, neutrophils, eosinophils, basophils and mastcells) to enhance or inhibit effector function. Many variations of suchmolecules have been described (see, e.g., United States PatentPublication Nos. 2015/0175697; 2014/0255407; 2014/0099318; 2013/0295121;2010/0174053; 2009/0060910; 2007/0004909; European Patent PublicationNos. EP 2714079; EP 2601216; EP 2376109; EP 2158221; EP 1868650; and PCTPublication Nos. WO 2012/162068; WO 2012/018687; WO 2010/080538; WO2006/113665), and are provided herein.

Recently, trivalent structures incorporating two Diabody-Type BindingDomains and one Non-Diabody-type Domain, and an Fc Domain have beendescribed (see, e.g., PCT Publication Nos. WO 2015/184207 and WO2015/184203). Such trivalent Binding Molecules may be utilized togenerate monospecific, bispecific or trispecific molecules as providedin more detail below. The ability to bind three different epitopesprovides enhanced capabilities.

Alternative constructs are known in the art for applications where abispecific or tetravalent molecule is desirable but an Fc is notrequired including, but not limited to, Bispecific T-cell Engagermolecules, also referred to as “BiTEs” (see, e.g., PCT Publication Nos:WO 1993/11161; and WO 2004/106381) and tetravalent tandem antibodies,also referred to as “TandAbs” (see, e.g. United States PatentPublication No: 2011-0206672; European Patent Publication No. EP2371866, and; PCT Publication Nos. WO 1999/057150, WO 2003/025018, andWO 2013/013700). BiTEs are formed from a single polypeptide chaincomprising tandem linked scFvs, while TandAbs are formed by thehomo-dimerization of two identical polypeptide chains, each possessing aVH1, VL2, VH2, and VL2 Domain.

The ability to produce multispecific Binding Molecules (e.g., bispecificantibodies, bispecific diabodies, trivalent molecules, etc.) has led totheir use (in “trans”) to co-ligate two cells together, for example, byco-ligating receptors that are present on the surface of different cells(e.g., cross-linking cytotoxic T-cells to target cells, such as cancercells or pathogen-infected cells, that express a Disease Antigen)(Staerz et al. (1985) “Hybrid Antibodies Can Target Sites For Attack ByT Cells,” Nature 314:628-631, and Holliger et al. (1996) “SpecificKilling Of Lymphoma Cells By Cytotoxic T-Cells Mediated By A BispecificDiabody,” Protein Eng. 9:299-305; Marvin et al. (2005) “RecombinantApproaches To IgG-Like Bispecific Antibodies,” Acta Pharmacol. Sin.26:649-658; Sloan et al. (2015) “Targeting HIV Reservoir in Infected CD4T Cells by Dual-Affinity Re-targeting Molecules (DARTs) that Bind HIVEnvelope and Recruit Cytotoxic T Cells,” PLoS Pathog 11(11): e1005233.doi:10.1371/journal.ppat.1005233)). Alternatively (or additionally),multispecific molecules can be used (in “cis”) to co-ligate molecules,such as receptors, etc., that are present on the surface of the samecell. Co-ligation of different cells and/or receptors is useful tomodulate effector functions and/or immune cell signaling. Multispecificmolecules (e.g., bispecific diabodies) comprising Epitope-BindingDomains may be directed to a surface determinant of any immune cell suchas CD2, CD3, CD8, CD16, TCR, the Natural Killer Group 2, Member DReceptor (NKG2D), etc., which are expressed on T lymphocytes, NaturalKiller (NK) cells, Antigen-Presenting Cells or other mononuclear cells.In particular, Epitope-Binding Domains directed to a cell surfacereceptor that is present on immune effector cells, are useful in thegeneration of multispecific Binding Molecules capable of mediatingredirected cell killing.

The present invention provides Binding Molecules that are capable ofmediating the redirected killing of a target cell (e.g., a cancer cellor a pathogen-infected cell, etc.) expressing a Disease Antigen (“DA”).Such Binding Molecules are capable of binding a “first epitope” and a“second epitope,” wherein one of such epitopes is an epitope of CD3 andthe other of such epitopes is an epitope of a Disease Antigen. It isirrelevant whether a particular epitope is designated as the first vs.the second epitope; such notation having relevance only with respect tothe presence and orientation of the domains of the polypeptide chains ofthe Binding Molecules of the present invention. Thus, the bispecificmolecules of the present invention comprise “VL_(CD3)”/“VH_(CD3)”Domains that are capable of binding an epitope of CD3, and“VL_(DA)”/“VH_(DA)” Domains that are capable of binding an epitope of aDisease Antigen. The instant invention particular encompasses bispecificdiabodies, bispecific scFvs, BiTEs, antibodies, TandAbs, and trivalentBinding Molecules produced using any of the methods provided herein.

A. Bispecific Diabodies Lacking Fc Domains

In one embodiment, the DA×CD3 Binding Molecule of the invention arebispecific diabodies and comprises domains capable of binding both afirst and a second epitope, but will lack an Fc Domain, and thus will beunable to bind FcγR molecules via an Fc-FcγR interaction. The firstpolypeptide chain of such an embodiment of bispecific diabodiescomprises, in the N-terminal to C-terminal direction: an N-terminus, theVL Domain of a monoclonal antibody capable of binding either the firstor second epitope (i.e., either VL_(CD3) or VL_(DA)), a firstintervening spacer peptide (Linker 1), a VH Domain of a monoclonalantibody capable of binding the epitope of the Disease Antigen (if suchfirst polypeptide chain contains VL_(CD3)) or a VH Domain of amonoclonal antibody capable of binding CD3 (if such first polypeptidechain contains VL_(DA)), a second intervening spacer peptide (Linker 2)optionally containing a cysteine residue, a Heterodimer-Promoting Domainand a C-terminus (FIGS. 1A-1B).

The second polypeptide chain of this embodiment of bispecific diabodiescomprises, in the N-terminal to C-terminal direction: an N-terminus, theVL Domain of a monoclonal antibody capable of binding the first orsecond epitope (i.e., VL_(CD3) or VL_(DA), and being the VL Domain notselected for inclusion in the first polypeptide chain of the diabody),an intervening spacer peptide (Linker 1), a VH Domain of a monoclonalantibody capable of binding either the first or second epitope (i.e.,VH_(CD3) or VH_(DA), and being the VH Domain not selected for inclusionin the first polypeptide chain of the diabody), a second interveningspacer peptide (Linker 2) optionally containing a cysteine residue, aHeterodimer-Promoting Domain and a C-terminus (FIGS. 1A-1B). Theemployed VL and VH Domains specific for a particular epitope arepreferably obtained or derived from the same monoclonal antibody.However, such domains may be derived from different monoclonalantibodies provided that they associate to form a functional bindingsite capable of immunospecifically binding such epitope. Such differentantibodies are referred to herein as being “corresponding” antibodies.

The VL Domain of the first polypeptide chain interacts with the VHDomain of the second polypeptide chain to form a first functionalEpitope-Binding Domain that is specific for one of the epitopes (e.g.,the first epitope). Likewise, the VL Domain of the second polypeptidechain interacts with the VH Domain of the first polypeptide chain inorder to form a second functional Epitope-Binding Domain that isspecific for the other epitope (i.e., the second epitope). Thus, theselection of the VL and VH Domains of the first and second polypeptidechains is “coordinated,” such that the two polypeptide chains of thediabody collectively comprise VL and VH Domains capable of binding boththe first epitope and the second epitope (i.e., they collectivelycomprise VL_(CD3)/VH_(CD3) and VL_(DA)/VH_(DA)).

Most preferably, the length of the intervening spacer peptide (i.e.,“Linker 1,” which separates such VL and VH Domains) is selected tosubstantially or completely prevent the VL and VH Domains of thepolypeptide chain from binding one another (for example consisting offrom 0, 1, 2, 3, 4, 5, 6, 7, 8 or 9 intervening Linker amino acidresidues). Thus, the VL and VH Domains of the first polypeptide chainare substantially or completely incapable of binding one another.Likewise, the VL and VH Domains of the second polypeptide chain aresubstantially or completely incapable of binding one another. Apreferred intervening spacer peptide (Linker 1) has the sequence (SEQ IDNO:16): GGGSGGGG.

The length and composition of the second intervening spacer peptide(“Linker 2”) is selected based on the choice of one or more polypeptidedomains that promote such dimerization (i.e., a “Heterodimer-PromotingDomain”). Typically, the second intervening spacer peptide (Linker 2)will comprise 3-20 amino acid residues. In particular, where theemployed Heterodimer-Promoting Domain(s) do/does not comprise a cysteineresidue a cysteine-containing second intervening spacer peptide (Linker2) is utilized. A cysteine-containing second intervening spacer peptide(Linker 2) will contain 1, 2, 3 or more cysteines. A preferredcysteine-containing spacer peptide (Linker 2) has the sequence GGCGGG(SEQ ID NO:17). Alternatively, Linker 2 does not comprise a cysteine(e.g., GGG, GGGS (SEQ ID NO:18), LGGGSG (SEQ ID NO:19), GGGSGGGSGGG (SEQID NO:20), ASTKG (SEQ ID NO:21), LEPKSS (SEQ ID NO:22), APSSS (SEQ IDNO:23), etc.) and a cysteine-containing Heterodimer-Promoting Domain, asdescribed below is used. Optionally, both a cysteine-containing Linker 2and a cysteine-containing Heterodimer-Promoting Domain are used.

The Heterodimer-Promoting Domains may comprise or consist of GVEPKSC(SEQ ID NO:24) or VEPKSC (SEQ ID NO:25) or AEPKSC (SEQ ID NO:26) on onepolypeptide chain and GFNRGEC (SEQ ID NO:27) or FNRGEC (SEQ ID NO:28) onthe other polypeptide chain (US2007/0004909).

In a preferred embodiment, the Heterodimer-Promoting Domains willcomprise tandemly repeated coil domains of opposing charge for example,an “E-coil” Heterodimer-Promoting Domain (SEQ ID NO:29:EVAALEK-EVAALEK-EVAALEK-EVAALEK), whose glutamate residues will form anegative charge at pH 7, or a “K-coil” Heterodimer-Promoting Domain (SEQID NO:30: KVAALKE-KVAALKE-KVAALKE-KVAALKE), whose lysine residues willform a positive charge at pH 7. The presence of such charged domainspromotes association between the first and second polypeptides, and thusfosters heterodimer formation. Heterodimer-Promoting Domains thatcomprise modifications of the above-described E-coil and K-coilsequences so as to include one or more cysteine residues may beutilized. The presence of such cysteine residues permits the coilpresent on one polypeptide chain to become covalently bonded to acomplementary coil present on another polypeptide chain, therebycovalently bonding the polypeptide chains to one another and increasingthe stability of the diabody. Examples of such particularly preferredare Heterodimer-Promoting Domains include a Modified E-Coil having theamino acid sequence EVAA

EK-EVAALEK-EVAALEK-{right arrow over (E)}VAALEK (SEQ ID NO:31), and amodified K-coil having the amino acid sequence KVAA

KE-KVAALKE-KVAALKE-KVAALKE (SEQ ID NO:32).

As disclosed in WO 2012/018687, in order to improve the in vivopharmacokinetic properties of diabodies, a diabody may be modified tocontain a polypeptide portion of a serum-binding protein at one or moreof the termini of the diabody. Most preferably, such polypeptide portionof a serum-binding protein will be installed at the C-terminus of apolypeptide chain of the diabody. Albumin is the most abundant proteinin plasma and has a half-life of 19 days in humans. Albumin possessesseveral small molecule binding sites that permit it to non-covalentlybind other proteins and thereby extend their serum half-lives. TheAlbumin-Binding Domain 3 (ABD3) of protein G of Streptococcus strainG148 consists of 46 amino acid residues forming a stable three-helixbundle and has broad albumin-binding specificity (Johansson, M. U. etal. (2002) “Structure, Specificity, And Mode Of Interaction ForBacterial Albumin-Binding Modules,” J. Biol. Chem. 277(10):8114-8120).Thus, a particularly preferred polypeptide portion of a serum-bindingprotein for improving the in vivo pharmacokinetic properties of adiabody is the Albumin-Binding Domain (ABD) from streptococcal proteinG, and more preferably, the Albumin-Binding Domain 3 (ABD3) of protein Gof Streptococcus strain G148 (SEQ ID NO:33):

LAEAKVLANR ELDKYGVSDY YKNLINNAKT VEGVKALIDE ILAALP.

As disclosed in WO 2012/162068 (herein incorporated by reference),“deimmunized” variants of SEQ ID NO:33 have the ability to attenuate oreliminate MHC class II binding. Based on combinational mutation results,the following combinations of substitutions are considered to bepreferred substitutions for forming such a deimmunized ABD: 66D/70S+71A;66S/70S+71A; 66S/70S+79A; 64A/65A/71A; 64A/65A/71A+66S; 64A/65A/71A+66D;64A/65A/71A+66E; 64A/65A/79A+66S; 64A/65A/79A+66D; 64A/65A/79A+66E.Variant ABDs having the modifications L64A, I65A and D79A or themodifications N66S, T70S and D79A. Variant deimmunized ABD having theamino acid sequence:

(SEQ ID NO: 34) LAEAKVLANR ELDKYGVSDY YKNLI D ₆₆NAK S ₇₀  A ₇₁EGVKALIDEILAALP, or the amino acid sequence: (SEQ ID NO: 35)LAEAKVLANR ELDKYGVSDY YKN A ₆₄ A ₆₅NNAKT VEGVKALI A ₇₉E ILAALP, orthe amino acid sequence: (SEQ ID NO: 36) LAEAKVLANR ELDKYGVSDY YKNLI S₆₆NAK S ₇₀ VEGVKALI A ₇₉E ILAALP,are particularly preferred as such deimmunized ABD exhibit substantiallywild-type binding while providing attenuated MHC class II binding. Thus,the first polypeptide chain of such a diabody having an ABD contains athird Linker (Linker 3) preferably positioned C-terminally to the E-coil(or K-coil) Domain of such polypeptide chain so as to intervene betweenthe E-coil (or K-coil) Domain and the ABD (which is preferably adeimmunized ABD). A preferred sequence for such Linker 3 is SEQ IDNO:18: GGGS.

B. Diabodies Comprising Fc Domains

One embodiment of the present invention relates to multispecificdiabodies (e.g., bispecific, trispecific, tetraspecific, etc.) thatcomprise an Fc Domain and that are capable of simultaneously binding anepitope of CD3 and an epitope of a Disease Antigen. The Fc Domain ofsuch molecules may be of any isotype (e.g., IgG1, IgG2, IgG3, or IgG4).The molecules may further comprise a CH1 Domain and/or a Hinge Domain.When present, the CH1 Domain and/or Hinge Domain may be of any isotype(e.g., IgG1, IgG2, IgG3, or IgG4), and is preferably of the same isotypeas the desired Fc Domain.

The addition of an IgG CH2-CH3 Domain to one or both of the diabodypolypeptide chains, such that the complexing of the diabody chainsresults in the formation of an Fc Domain, increases the biologicalhalf-life and/or alters the valency of the diabody. Such diabodiescomprise, two or more polypeptide chains whose sequences permit thepolypeptide chains to covalently bind each other to form a covalentlyassociated diabody that is capable of simultaneously binding the firstepitope and the second epitope. Incorporating an IgG CH2-CH3 Domainsonto both of the diabody polypeptides will permit a two-chain bispecificFc Domain-containing diabody to form (FIG. 2 ).

Alternatively, incorporating IgG CH2-CH3 Domains onto only one of thediabody polypeptides will permit a more complex four-chain bispecific FcDomain-containing diabody to form (FIGS. 3A-3C). FIG. 3C shows arepresentative four-chain diabody possessing the Constant Light (CL)Domain and the Constant Heavy CH1 Domain, however fragments of suchdomains as well as other polypeptides may alternatively be employed(see, e.g., FIGS. 3A and 3B, United States Patent Publication Nos.2013-0295121; 2010-0174053 and 2009-0060910; European Patent PublicationNo. EP 2714079; EP 2601216; EP 2376109; EP 2158221 and PCT PublicationNos. WO 2012/162068; WO 2012/018687; WO 2010/080538). Thus, for example,in lieu of the CH1 Domain, one may employ a peptide having the aminoacid sequence GVEPKSC (SEQ ID NO:24), VEPKSC (SEQ ID NO:25), or AEPKSC(SEQ ID NO:26), derived from the Hinge Domain of a human IgG, and inlieu of the CL Domain, one may employ the C-terminal 6 amino acids ofthe human kappa Light Chain, GFNRGEC (SEQ ID NO:27) or FNRGEC (SEQ IDNO:28). A representative peptide containing four-chain diabody is shownin FIG. 3A. Alternatively, or in addition, one may employ a peptidecomprising tandem coil domains of opposing charge such as the “E-coil”helical domains (SEQ ID NO:29: EVAALEK-EVAALEK-EVAALEK-EVAALEK or SEQ IDNO:31: EVAA

EK-EVAALEK-EVAALEK-EVAALEK); and the “K-coil” domains (SEQ ID NO:30:KVAALKE-KVAALKE-KVAALKE-KVAALKE or SEQ ID NO:32: KVAA

KE-KVAALKE-KVAALKE-KVAALKE). A representative coil domain containingfour-chain diabody is shown in FIG. 3B.

Fc Domain-containing diabody molecules of the present invention mayinclude additional intervening spacer peptides (Linkers), generally suchLinkers will be incorporated between a Heterodimer-Promoting Domain(e.g., an E-coil or K-coil) and a CH2-CH3 Domain and/or between aCH2-CH3 Domain and a Variable Domain (i.e., VH or VL). Typically, theadditional Linkers will comprise 3-20 amino acid residues and mayoptionally contain all or a portion of an IgG Hinge Domain (preferably acysteine-containing portion of an IgG Hinge Domain possessing 1, 2, 3 ormore cysteine residues). Linkers that may be employed in the bispecificFc Domain-containing diabody molecules of the present invention include:GGGS (SEQ ID NO:18), LGGGSG (SEQ ID NO:19), GGGSGGGSGGG (SEQ ID NO:20),ASTKG (SEQ ID NO:21), LEPKSS (SEQ ID NO:22), APSSS (SEQ ID NO:23),APSSSPME (SEQ ID NO:37), VEPKSADKTHTCPPCP (SEQ ID NO:38),LEPKSADKTHTCPPCP (SEQ ID NO:39), DKTHTCPPCP (SEQ ID NO:40), the scFvLinker: GGGGSGGGGSGGGGS (SEQ ID NO:41); the “long” Linker: GGGGSGGGSGGG(SEQ ID NO:42), GGC, and GGG. LEPKSS (SEQ ID NO:22) may be used in lieuof GGG or GGC for ease of cloning. Additionally, the amino acids GGG, orLEPKSS (SEQ ID NO:22) may be immediately followed by DKTHTCPPCP (SEQ IDNO:40) to form the alternate Linkers: GGGDKTHTCPPCP (SEQ ID NO:43); andLEPKSSDKTHTCPPCP (SEQ ID NO:44). Bispecific Fc Domain-containingmolecules of the present invention may incorporate an IgG Hinge Domainin addition to or in place of a Linker. Exemplary Hinge Domains include:EPKSCDKTHTCPPCP (SEQ ID NO:5) from IgG1, ERKCCVECPPCP (SEQ ID NO:6) fromIgG2, ELKTPLGDTT HTCPRCPEPKSCDTPPPCPRCPEPKSCDTPPPCPRCPEPKSCDTPPPCPRCP(SEQ ID NO:7) from IgG3, ESKYGPPCPSCP (SEQ ID NO:8) from IgG4, andESKYGPPCPPCP (SEQ ID NO:9) an IgG4 Hinge variant comprising astabilizing S228P substitution (as numbered by the EU index as set forthin Kabat) to reduce strand exchange.

As provided in FIG. 3A-3C, Fc Domain-containing diabodies of theinvention may comprise four chains. The first and third polypeptidechains of such a diabody contain three domains: (i) a VL1-containingDomain, (ii) a VH2-containing Domain, (iii) a Heterodimer-PromotingDomain, and (iv) a Domain containing a CH2-CH3 sequence. The second andfourth polypeptide chains contain: (i) a VL2-containing Domain, (ii) aVH1-containing Domain, and (iii) a Heterodimer-Promoting Domain, wherethe Heterodimer-Promoting Domains promote the dimerization of thefirst/third polypeptide chains with the second/fourth polypeptidechains. The VL and/or VH Domains of the third and fourth polypeptidechains, and VL and/or VH Domains of the first and second polypeptidechains may be the same or different so as to permit tetravalent bindingthat is either monospecific, bispecific or tetraspecific. The notation“VL3” and “VH3” denote respectively, the Light Chain Variable Domain andVariable Heavy Chain Domain that bind a “third” epitope of such diabody.Similarly, the notation “VL4” and “VH4” denote respectively, the LightChain Variable Domain and Variable Heavy Chain Domain that bind a“fourth” epitope of such diabody. The general structure of thepolypeptide chains of a representative four-chain bispecific FcDomain-containing diabodies of invention is provided in Table 1:

TABLE 1 Bispecific 2^(nd )Chain NH₂—VL2—VH1—HPD—COOH 1^(st )ChainNH₂—VL1—VH2—HPD—CH2—CH3—COOH 1^(st )Chain NH₂—VL1—VH2—HPD—CH2—CH3—COOH2^(nd )Chain NH₂—VL2—VH1—HPD—COOH Tetraspe- 2^(nd )ChainNH₂—VL2—VH1—HPD—COOH cific 1^(st )Chain NH₂—VL1—VH2—HPD—CH2—CH3—COOH3^(rd )Chain NH₂—VL3—VH4—HPD—CH2—CH3—COOH 4^(th )ChainNH₂—VL4—VH3—HPD—COOH HPD = Heterodimer-Promoting Domain

In a specific embodiment, diabodies of the present invention arebispecific, tetravalent (i.e., possess four Epitope-Binding Domains),Fc-containing diabodies that are composed of four total polypeptidechains (FIGS. 3A-3C). The bispecific, tetravalent, Fc-containingdiabodies of the invention comprise two first Epitope-Binding Domainsand two second Epitope-Binding Domains.

In a further embodiment, the Fc Domain-containing diabodies of thepresent invention may comprise three polypeptide chains. The firstpolypeptide of such a diabody contains three domains: (i) aVL1-containing Domain, (ii) a VH2-containing Domain and (iii) a Domaincontaining a CH2-CH3 sequence. The second polypeptide of such a diabodycontains: (i) a VL2-containing Domain, (ii) a VH1-containing Domain and(iii) a Domain that promotes heterodimerization and covalent bondingwith the diabody's first polypeptide chain. The third polypeptide ofsuch a diabody comprises a CH2-CH3 sequence. Thus, the first and secondpolypeptide chains of such a diabody associate together to form aVL1/VH1 Epitope-Binding Domain that is capable of binding either thefirst or second epitope, as well as a VL2/VH2 Epitope-Binding Domainthat is capable of binding the other of such epitopes. The first andsecond polypeptides are bonded to one another through a disulfide bondinvolving cysteine residues in their respective Third Domains. Notably,the first and third polypeptide chains complex with one another to forman Fc Domain that is stabilized via a disulfide bond. Such bispecificdiabodies have enhanced potency. FIGS. 4A and 4B illustrate thestructures of such diabodies. Such Fc Domain-containing diabodies mayhave either of two orientations (Table 2):

TABLE 2 First 3^(rd )Chain NH₂—CH2—CH3—COOH Orien- 1^(st )ChainNH₂—VL1—VH2—HPD—CH2—CH3—COOH tation 2^(nd )Chain NH₂—VL2—VH1—HPD—COOHSecond 3^(rd )Chain NH₂—CH2—CH3—COOH Orien- 1^(st )ChainNH₂—CH2—CH3—VL1—VH2—HPD—COOH tation 2^(nd )Chain NH₂—VL2—VH1—HPD—COOHHPD = Heterodimer-Promoting Domain

In a specific embodiment, diabodies of the present invention arebispecific, bivalent (i.e., possess two Epitope-Binding Domains),Fc-containing diabodies that are composed of three total polypeptidechains (FIGS. 4A-4B). The bispecific, bivalent Fc-containing diabodiesof the invention comprise one Epitope-Binding Domain immunospecific foreither the first or second epitope, as well as a VL2/VH2 Epitope-BindingDomain that is capable of binding the other of such epitopes.

In a further embodiment, the Fc Domain-containing diabodies may comprisea total of five polypeptide chains. In a particular embodiment, two ofthe five polypeptide chains have the same amino acid sequence. The firstpolypeptide chain of such a diabody contains: (i) a VH1-containingDomain, (ii) a CH1-containing Domain, and (iii) a Domain containing aCH2-CH3 sequence. The first polypeptide chain may be the Heavy Chain ofan antibody that contains a VH1 and a Heavy Chain constant region. Thesecond and fifth polypeptide chains of such a diabody contain: (i) aVL1-containing Domain, and (ii) a CL-containing Domain. The secondand/or fifth polypeptide chains of such a diabody may be Light Chains ofan antibody that contains a VL1 complementary to the VH1 of thefirst/third polypeptide chain. The first, second and/or fifthpolypeptide chains may be isolated from a naturally occurring antibody.Alternatively, they may be constructed recombinantly. The thirdpolypeptide chain of such a diabody contains: (i) a VH1-containingDomain, (ii) a CH1-containing Domain, (iii) a Domain containing aCH2-CH3 sequence, (iv) a VL2-containing Domain, (v) a VH3-containingDomain and (vi) a Heterodimer-Promoting Domain, where theHeterodimer-Promoting Domains promote the dimerization of the thirdchain with the fourth chain. The fourth polypeptide of such diabodiescontains: (i) a VL3-containing Domain, (ii) a VH2-containing Domain and(iii) a Domain that promotes heterodimerization and covalent bondingwith the diabody's third polypeptide chain.

Thus, the first and second, and the third and fifth, polypeptide chainsof such diabodies associate together to form two VL1/VH1 Epitope-BindingDomains capable of binding a first epitope. The third and fourthpolypeptide chains of such diabodies associate together to form aVL2/VH2 Epitope-Binding Domain that is capable of binding a secondepitope, as well as a VL3/VH3 binding site that is capable of binding athird epitope. The first and third polypeptides are bonded to oneanother through a disulfide bond involving cysteine residues in theirrespective constant regions. Notably, the first and third polypeptidechains complex with one another to form an Fc Domain. Such multispecificdiabodies have enhanced potency. FIG. 5 illustrates the structure ofsuch diabodies. It will be understood that the VL1/VH1, VL2/VH2, andVL3/VH3 Domains may be the same or different so as to permit bindingthat is monospecific, bispecific or trispecific.

The VL and VH Domains of the polypeptide chains are selected so as toform VL/VH binding sites specific for a desired epitope. The VL/VHbinding sites formed by the association of the polypeptide chains may bethe same or different so as to permit tetravalent binding that ismonospecific, bispecific, trispecific or tetraspecific. In particular,the VL and VH Domains maybe selected such that a multivalent diabody maycomprise two binding sites for a first epitope and two binding sites fora second epitope, or three binding sites for a first epitope and onebinding site for a second epitope, or two binding sites for a firstepitope, one binding site for a second epitope and one binding site fora third epitope (as depicted in FIG. 5 ). The general structure of thepolypeptide chains of representative five-chain Fc Domain-containingdiabodies of invention is provided in Table 3:

TABLE 3 Bispecific 2^(nd )Chain NH₂—VL1—CL—COOH (2 × 2) 1^(st )ChainNH₂—VH1—CH1—CH2—CH3—COOH 3^(rd )ChainNH₂—VH1—CH1—CH2—CH3—VL2—VH2—HPD—COOH 5^(nd )Chain NH₂—VL1—CL—COOH4^(th )Chain NH₂—VL2—VH2—HPD—COOH Bispecific 2^(nd )ChainNH₂—VL1—CL—COOH (3 × 1) 1^(st )Chain NH₂—VH1—CH1—CH2—CH3—COOH3^(rd )Chain NH₂—VH1—CH1—CH2—CH3—VL1—VH2—HPD—COOH 5^(nd )ChainNH₂—VL1—CL—COOH 4^(th )Chain NH₂—VL2—VH1—HPD—COOH Trispecific2^(nd )Chain NH₂—VL1—CL—COOH (2 × 1 × 1) 1^(st )ChainNH₂—VH1—CH1—CH2—CH3—COOH 3^(rd )ChainNH₂—VH1—CH1—CH2—CH3—VL2—VH3—HPD—COOH 5^(nd )Chain NH₂—VL1—CL—COOH4^(th )Chain NH₂—VL3—VH2—HPD—COOH HPD = Heterodimer-Promoting Domain

In a specific embodiment, diabodies of the present invention arebispecific, tetravalent (i.e., possess four Epitope-Binding Domains),Fc-containing diabodies that are composed of five total polypeptidechains having two Epitope-Binding Domains immunospecific for the firstepitope, and two Epitope-Binding Domains specific for the secondepitope. In another embodiment, the bispecific, tetravalent,Fc-containing diabodies of the invention comprise three Epitope-BindingDomains immunospecific for the first epitope and one Epitope-BindingDomain specific for the second epitope. As provided above, the VL and VHDomains may be selected to permit trispecific binding. Accordingly, theinvention also encompasses trispecific, tetravalent, Fc-containingdiabodies. The trispecific, tetravalent, Fc-containing diabodies of theinvention comprise two Epitope-Binding Domains immunospecific for thefirst epitope, one Epitope-Binding Domain immunospecific for the secondmolecule, and one Epitope-Binding Domain immunospecific for the thirdepitope.

In traditional immune function, the interaction of antibody-antigencomplexes with cells of the immune system results in a wide array ofresponses, ranging from effector functions such as antibody-dependentcytotoxicity, mast cell degranulation, and phagocytosis toimmunomodulatory signals such as regulating lymphocyte proliferation andantibody secretion. All of these interactions are initiated through thebinding of the Fc Domain of antibodies or immune complexes tospecialized cell surface receptors on hematopoietic cells. The diversityof cellular responses triggered by antibodies and immune complexesresults from the structural heterogeneity of the three Fc Receptors:FcγRI (CD64), FcγRII (CD32), and FcγRIII (CD16). FcγRI (CD64), FcγRIIA(CD32A) and FcγRIII (CD16) are activating (i.e., immune systemenhancing) receptors; FcγRIIB (CD32B) is an inhibiting (i.e., immunesystem dampening) receptor. In addition, interaction with the neonatalFc Receptor (FcRn) mediates the recycling of IgG molecules from theendosome to the cell surface and release into the blood. The amino acidsequence of exemplary wild-type IgG1 (SEQ ID NO:10), IgG2 (SEQ IDNO:11), IgG3 (SEQ ID NO:12), and IgG4 (SEQ ID NO:13) are presentedabove.

Modification of the Fc Domain may lead to an altered phenotype, forexample altered serum half-life, altered stability, alteredsusceptibility to cellular enzymes or altered effector function. It maytherefore be desirable to modify an Fc Domain-containing bindingmolecule of the present invention with respect to effector function, forexample, so as to enhance the effectiveness of such molecule in treatingcancer. Reduction or elimination of Fc Domain-mediated effector functionis desirable in certain cases, for example in the case of antibodieswhose mechanism of action involves blocking or antagonism, but notkilling of the cells bearing a target antigen. Increased effectorfunction is generally desirable when directed to undesirable cells, suchas tumor and foreign cells, where the FcγRs are expressed at low levels,for example, tumor-specific B cells with low levels of FcγRIIB (e.g.,non-Hodgkin's lymphoma, CLL, and Burkitt's lymphoma). Molecules of theinvention possessing such conferred or altered effector functionactivity are useful for the treatment and/or prevention of a disease,disorder or infection in which an enhanced efficacy of effector functionactivity is desired.

Accordingly, in certain embodiments, the Fc Domain of the FcDomain-containing molecules of the present invention may be anengineered variant Fc Domain. Although the Fc Domain of the bispecificFc Domain-containing molecules of the present invention may possess theability to bind one or more Fc Receptors (e.g., FcγR(s)), morepreferably such variant Fc Domain have altered binding FcγRIA (CD64),FcγRIIA (CD32A), FcγRIIB (CD32B), FcγRIIIA (CD16a) or FcγRIIIB (CD16b)(relative to the binding exhibited by a wild-type Fc Domain), e.g., willhave enhanced binding an activating receptor and/or will havesubstantially reduced or no ability to bind inhibitory receptor(s).Thus, the Fc Domain of the Fc Domain-containing molecules of the presentinvention may include some or all of the CH2 Domain and/or some or allof the CH3 Domain of a complete Fc Domain, or may comprise a variant CH2and/or a variant CH3 sequence (that may include, for example, one ormore insertions and/or one or more deletions with respect to the CH2 orCH3 Domains of a complete Fc Domain). Such Fc Domains may comprisenon-Fc polypeptide portions, or may comprise portions of non-naturallycomplete Fc Domains, or may comprise non-naturally occurringorientations of CH2 and/or CH3 Domains (such as, for example, two CH2Domains or two CH3 Domains, or in the N-terminal to C-terminaldirection, a CH3 Domain linked to a CH2 Domain, etc.).

Fc Domain modifications identified as altering effector function areknown in the art, including modifications that increase bindingactivating receptors (e.g., FcγRIIA (CD16A) and reduce bindinginhibitory receptors (e.g., FcγRIIB (CD32B) (see, e.g., Stavenhagen, J.B. et al. (2007) “Fc Optimization Of Therapeutic Antibodies EnhancesTheir Ability To Kill Tumor Cells In Vitro And Controls Tumor ExpansionIn Vivo Via Low-Affinity Activating Fcgamma Receptors,” Cancer Res.57(18):8882-8890). Table 4 lists exemplary single, double, triple,quadruple and quintuple substitutions (numbering (according to the EUindex) and substitutions are relative to the amino acid sequence of SEQID NO:10 as presented above) of exemplary modification that increasebinding activating receptors and/or reduce binding inhibitory receptors.

TABLE 4 Variations of Preferred Activating Fc Domains† Single-SiteVariations F243L R292G D270E R292P Y300L P396L Double-Site VariationsF243L and R292P F243L and Y300L F243L and P396L R292P and Y300L D270Eand P396L R292P and V305I P396L and Q419H P247L and N421K R292P andP396L Y300L and P396L R255L and P396L R292P and P305I K392T and P396LTriple-Site Variations F243L, P247L and N421K P247L, D270E and N421KF243L, R292P and Y300L R255L, D270E and P396L F243L, R292P and V305ID270E, G316D and R416G F243L, R292P and P396L D270E, K392T and P396LF243L, Y300L and P396L D270E, P396L and Q419H V284M, R292L and K370NR292P, Y300L and P396L Quadruple-Site Variations L234F, F243L, R292P andY300L F243L, P247L, D270E and N421K L234F, F243L, R292P and Y300L F243L,R255L, D270E and P396L L235I, F243L, R292P and Y300L F243L, D270E, G316DandR416G L235Q, F243L, R292P and Y300L F243L, D270E, K392T and P396LP247L, D270E, Y300L and N421K F243L, R292P, Y300L, and P396L R255L,D270E, R292G and P396L F243L, R292P, V305I and P396L R255L, D270E, Y300Land P396L F243L, D270E, P396L and Q419H D270E, G316D, P396L and R416GQuintuple-Site Variations L235V, F243L, R292P, Y300L and P396L F243L,R292P, V305I, Y300L and P396L L235P, F243L, R292P, Y300L and P396L†numbering is according to the EU index as in Kabat

Exemplary variants of human IgG1 Fe Domains with reduced binding CD32Band/or increased binding CD16A contain F243L, R292P, Y300L, V305I orP396L substitutions, wherein the numbering is that of the EU index as inKabat. These amino acid substitutions may be present in a human IgG1 FcDomain in any combination. In one embodiment, the variant human IgG1 FcDomain contains a F243L, R292P and Y300L substitution. In anotherembodiment, the variant human IgG1 Fc Domain contains a F243L, R292P,Y300L, V305I and P396L substitution.

In certain embodiments, it is preferred for the Fc Domains of the FcDomain-containing Binding Molecules of the present invention to exhibitdecreased (or substantially no) binding FcγRIA (CD64), FcγRIIA (CD32A),FcγRIIB (CD32B), FcγRIIA (CD16a) or FcγRIIIB (CD16b) (relative to thebinding exhibited by the wild-type IgG1 Fc Domain (SEQ ID NO:10)). In aspecific embodiment, the Fc Domain-containing Binding Molecules of thepresent invention comprise an IgG Fc Domain that exhibits reducedantibody-dependent cell-mediated cytotoxicity (ADCC) effector function.In a preferred embodiment, the CH2-CH3 Domains of such Binding Moleculesinclude any 1, 2, 3, or 4 of the substitutions: L234A, L235A, D265A,N297Q, and N297G, wherein the numbering is that of the EU index as inKabat. In another embodiment, the CH2-CH3 Domains contain an N297Qsubstitution, an N297G substitution, L234A and L235A substitutions or aD265A substitution, as these mutations abolish FcR binding.Alternatively, a CH2-CH3 Domain of a naturally occurring Fc Domain thatinherently exhibits decreased (or substantially no) binding FcγRIIIA(CD16a) and/or reduced effector function (relative to the binding andeffector function exhibited by the wild-type IgG1 Fc Domain (SEQ IDNO:10)) is utilized. In a specific embodiment, the Fc Domain-containingBinding Molecules of the present invention comprise an IgG2 Fc Domain(SEQ ID NO:11), an IgG3 Fc Domain (SEQ ID NO:12) or an IgG4 Fc Domain(SEQ ID NO:13). When an IgG4 Fc Domain is utilized, the instantinvention also encompasses the introduction of a stabilizing mutation,such as the Hinge Region S228P substitution described above (see, e.g.,SEQ ID NO:9). Since the N297G, N297Q, L234A, L235A and D265Asubstitutions abolish effector function, in circumstances in whicheffector function is desired, these substitutions would preferably notbe employed.

A preferred IgG1 sequence for the CH2 and CH3 Domains of the FcDomain-containing molecules of the present invention having reduced orabolished effector function will comprise the substitutions L234A/L235A(SEQ ID NO:45):

APE AA GGPSV FLFPPKPKDT LMISRTPEVT CVVVDVSHEDPEVKFNWYVD GVEVHNAKTK PREEQYNSTY RVVSVLTVLHQDWLNGKEYK CKVSNKALPA PIEKTISKAK GQPREPQVYTLPPSREEMTK NQVSLTCLVK GFYPSDIAVE WESNGQPENNYKTIPPVLDS DGSFFLYSKL TVDKSRWQQG NVFSCSVMHE ALHNHYTQKS LSLSPG X

-   -   wherein, X is a lysine (K) or is absent.

The serum half-life of proteins comprising Fc Domains may be increasedby increasing the binding affinity of the Fc Domain for FcRn. The term“half-life” as used herein means a pharmacokinetic property of amolecule that is a measure of the mean survival time of the moleculesfollowing their administration. Half-life can be expressed as the timerequired to eliminate fifty percent (50%) of a known quantity of themolecule from a subject's body (e.g., a human patient or other mammal)or a specific compartment thereof, for example, as measured in serum,i.e., circulating half-life, or in other tissues. In general, anincrease in half-life results in an increase in mean residence time(MRT) in circulation for the molecule administered.

In some embodiments, the Fc Domain-containing Binding Molecules of thepresent invention comprise a variant Fc Domain that comprises at leastone amino acid modification relative to a wild-type Fc Domain, such thatthe molecule has an increased half-life (relative to such molecule ifcomprising a wild-type Fc Domain). In some embodiments, the FcDomain-containing Binding Molecules of the present invention comprise avariant IgG Fc Domain that comprises a half-life extending amino acidsubstitution at one or more positions selected from the group consistingof 238, 250, 252, 254, 256, 257, 256, 265, 272, 286, 288, 303, 305, 307,308, 309, 311, 312, 317, 340, 356, 360, 362, 376, 378, 380, 382, 413,424, 428, 433, 434, 435, and 436, wherein the numbering is that of theEU index as in Kabat. Numerous mutations capable of increasing thehalf-life of an Fc Domain-containing molecule are known in the art andinclude, for example M252Y, S254T, T256E, and combinations thereof. Forexample, see the mutations described in U.S. Pat. Nos. 6,277,375,7,083,784; 7,217,797, 8,088,376; U.S. Publication Nos. 2002/0147311;2007/0148164; and PCT Publication Nos. WO 98/23289; WO 2009/058492; andWO 2010/033279, which are herein incorporated by reference in theirentireties.

In some embodiments, the Fc Domain-containing Binding Molecules of thepresent invention exhibiting enhanced half-life possess a variant FcDomain comprising substitutions at two or more of Fc Domain residues250, 252, 254, 256, 257, 288, 307, 308, 309, 311, 378, 428, 433, 434,435 and 436. In particular, two or more substitutions selected from:T250Q, M252Y, S254T, T256E, K288D, T307Q, V308P, A378V, M428L, N434A,H435K, and Y436I. In a specific embodiment, such molecules may possess avariant IgG Fc Domain comprising the substitution:

-   -   (A) M252Y, S254T and T256E;    -   (B) M252Y and S254T;    -   (C) M252Y and T256E;    -   (D) T250Q and M428L;    -   (E) T307Q and N434A;    -   (F) A378V and N434A;    -   (G) N434A and Y436I;    -   (H) V308P and N434A; or    -   (I) K288D and H435K.

In a preferred embodiment, an Fc Domain-containing binding molecule ofthe present invention possesses a variant IgG Fc Domain comprising any1, 2, or 3 of the substitutions: M252Y, S254T and T256E. The inventionfurther encompasses such Binding Molecules that possess a variant FcDomain comprising:

-   -   (A) one or more mutations which alter effector function and/or        FcγR binding; and    -   (B) one or more mutations which extend serum half-life.

An IgG1 sequence for the CH2 and CH3 Domains of the Fc Domain-containingmolecules of the present invention that provides an increased half-life(and that has a 10-fold increase in binding to both cynomolgus monkeyand human FcRn) (Dall'Acqua, W. F. et al. (2006) “Properties of HumanIgG1s Engineered for Enhanced Binding to the Neonatal Fc Receptor(FcRn),” J. Biol. Chem. 281(33):23514-23524) will comprise thesubstitutions M252Y/S254T/T256E (SEQ ID NO:46):

APELLGGPSV FLFPPKPKDT L Y I T R E PEVT CVVVDVSHEDPEVKFNWYVD GVEVHNAKTK PREEQYNSTY RVVSVLTVLHQDWLNGKEYK CKVSNKALPA PIEKTISKAK GQPREPQVYTLPPSREEMTK NQVSLTCLVK GFYPSDIAVE WESNGQPENNYKTTPPVLDS DGSFFLYSKL TVDKSRWQQG NVFSCSVMHE ALHNHYTQKS LSLSPG X

-   -   wherein, X is a lysine (K) or is absent.

An alternative IgG1 sequence for the CH2 and CH3 Domains of the FcDomain-containing molecules of the present invention combining thereduced or abolished effector function provided by the substitutionsL234A/L235A and the increased serum half-life provided by thesubstitutions M252Y/S254T/T256E is provided by SEQ ID NO: 47:

APE AA GGPSV FLFPPKPKDT L Y I T R E PEVT CVVVDVSHEDPEVKFNWYVD GVEVHNAKTK PREEQYNSTY RVVSVLTVLHQDWLNGKEYK CKVSNKALPA PIEKTISKAK GQPREPQVYTLPPSREEMTK NQVSLTCLVK GFYPSDIAVE WESNGQPENNYKTIPPVLDS DGSFFLYSKL TVDKSRWQQG NVFSCSVMHE ALHNHYTQKS LSLSPG X

-   -   wherein, X is a lysine (K) or is absent.

For certain antibodies, diabodies and trivalent Binding Molecules thatare desired to have Fc-Domain-containing polypeptide chains of differingamino acid sequence (e.g., whose Fc Domain-containing polypeptide chainsare desired to not be identical), it is desirable to reduce or preventhomodimerization from occurring between the CH2-CH3 Domains of identicalchains (e.g., two first polypeptide chains or between the CH2-CH3Domains of two third polypeptide chains). The CH2 and/or CH3 Domains ofsuch polypeptide chains need not be identical in sequence, andadvantageously are modified to foster complexing between the twopolypeptide chains. For example, an amino acid substitution (preferablya substitution with an amino acid comprising a bulky side group forminga “knob”, e.g., tryptophan) can be introduced into the CH2 or CH3 Domainsuch that steric interference will prevent interaction with a similarlymutated domain and will obligate the mutated domain to pair with adomain into which a complementary, or accommodating mutation has beenengineered, i.e., “the hole” (e.g., a substitution with glycine). Suchsets of mutations can be engineered into any pair of polypeptidescomprising CH2-CH3 Domains that forms an Fc Domain to fosterheterodimerization. Methods of protein engineering to favorheterodimerization over homodimerization are well-known in the art, inparticular with respect to the engineering of immunoglobulin-likemolecules, and are encompassed herein (see e.g., Ridgway et al. (1996)“‘Knobs-Into-Holes’ Engineering Of Antibody CH3 Domains For Heavy ChainHeterodimerization,” Protein Engr. 9:617-621, Atwell et al. (1997)“Stable Heterodimers From Remodeling The Domain Interface Of A HomodimerUsing A Phage Display Library,” J. Mol. Biol. 270: 26-35, and Xie et al.(2005) “A New Format Of Bispecific Antibody: Highly EfficientHeterodimerization, Expression And Tumor Cell Lysis,” J. Immunol.Methods 296:95-101; each of which is hereby incorporated herein byreference in its entirety).

A preferred knob is created by modifying an IgG Fc Domain to contain themodification T366W. A preferred hole is created by modifying an IgG FcDomain to contain the modification T366S, L368A and Y407V. To aid inpurifying a hole-bearing polypeptide chain homodimer from the finalbispecific heterodimeric Fc Domain-containing molecule, the protein Abinding site of the hole-bearing CH2 and CH3 Domains a polypeptide chainis preferably mutated by amino acid substitution at position 435(H435R). Thus, the hole-bearing polypeptide chain homodimer will notbind protein A, whereas the bispecific heterodimer will retain itsability to bind protein A via the protein A binding site on theknob-bearing polypeptide chain. In an alternative embodiment, thehole-bearing polypeptide chain may incorporate amino acid substitutionsat positions 434 and 435 (N434A/N435K).

A preferred IgG1 amino acid sequence for the CH2 and CH3 Domains of oneFc Domain-containing polypeptide chain of an Fc Domain-containingmolecule of the present invention will have the “knob-bearing” sequence(SEQ ID NO:48):

APE AA GGPSV FLFPPKPKDT LMISRTPEVT CVVVDVSHEDPEVKFNWYVD GVEVHNAKTK PREEQYNSTY RVVSVLTVLHQDWLNGKEYK CKVSNKALPA PIEKTISKAK GQPREPQVYT LPPSREEMTK NQVSL WCLVK GFYPSDIAVE WESNGQPENN YKTIPPVLDS DGSFFLYSKL TVDKSRWQQG NVFSCSVMHEALHNHYTQKS LSLSPG X

-   -   wherein X is a lysine (K) or is absent.

An alternative IgG1 amino acid sequence for the CH2 and CH3 Domains ofone Fc Domain-containing polypeptide chain of an Fc Domain-containingmolecule of the present invention having a M252Y/S254T/T256Esubstitution and a “knob-bearing” sequence is SEQ ID NO:49:

APE AA GGPSV FLFPPKPKDT L Y I T R E PEVT CVVVDVSHEDPEVKFNWYVD GVEVHNAKTK PREEQYNSTY RVVSVLTVLHQDWLNGKEYK CKVSNKALPA PIEKTISKAK GQPREPQVYT LPPSREEMTK NQVSL WCLVK GFYPSDIAVE WESNGQPENN YKTIPPVLDS DGSFFLYSKL TVDKSRWQQG NVFSCSVMHEALHNHYTQKS LSLSPG X

-   -   wherein X is a lysine (K) or is absent.

A preferred IgG1 amino acid sequence for the CH2 and CH3 Domains of theother Fc Domain-containing polypeptide chain of an Fc Domain-containingmolecule of the present invention will have the “hole-bearing” sequence(SEQ ID NO:50):

APE AA GGPSV FLFPPKPKDT LMISRTPEVT CVVVDVSHEDPEVKFNWYVD GVEVHNAKTK PREEQYNSTY RVVSVLTVLHQDWLNGKEYK CKVSNKALPA PIEKTISKAK GQPREPQVYT LPPSREEMTK NQVSL S C AVK GFYPSDIAVE WESNGQPENN YKTTPPVLDS DGSFFL V SKL TVDKSRWQQG NVFSCSVMHEALHN R YTQKS LSLSPG X

-   -   wherein X is a lysine (K) or is absent.

An alternative IgG1 amino acid sequence for the CH2 and CH3 Domains ofthe other Fc Domain-containing polypeptide chain of an FcDomain-containing molecule of the present invention having aM252Y/S254T/T256E substitution and a “hole-bearing” sequence is SEQ IDNO:51:

APE AA GGPSV FLFPPKPKDT L Y I T R E PEVT CVVVDVSHEDPEVKFNWYVD GVEVHNAKTK PREEQYNSTY RVVSVLTVLHQDWLNGKEYK CKVSNKALPA PIEKTISKAK GQPREPQVYT LPPSREEMTK NQVSL S C AVK GFYPSDIAVE WESNGQPENN YKTTPPVLDS DGSFEL V SKL TVDKSRWQQG NVFSCSVMHEALHNRYTQKS LSLSPG X

-   -   wherein X is a lysine (K) or is absent.

As will be noted, the CH2-CH3 Domains of SEQ ID NO:48, SEQ ID NO:49, SEQID NO:50 and SEQ ID NO:51 include a substitution at position 234 withalanine and 235 with alanine, and thus form an Fc Domain exhibitdecreased (or substantially no) binding FcγRIA (CD64), FcγRIIA (CD32A),FcγRIIB (CD32B), FcγRIIIA (CD16a) or FcγRIIIB (CD16b) (relative to thebinding exhibited by the wild-type Fc Domain (SEQ ID NO:10)). Theinvention also encompasses such CH2-CH3 Domains, which comprise thewild-type alanine residues, alternative and/or additional substitutionswhich modify effector function and/or FγR binding activity of the FcDomain. The invention also encompasses such CH2-CH3 Domains, whichfurther comprise one or more half-live extending amino acidsubstitutions. In particular, the invention encompasses suchhole-bearing and such knob-bearing CH2-CH3 Domains which furthercomprise the M252Y/S254T/T256E.

An IgG4 amino acid sequence for the CH2 and CH3 Domains of the firstpolypeptide chain of an Fc Domain-containing molecule of the presentinvention has enhanced serum half-life (relative to IgG1 CH2 and CH3Domains) due to its possession of Y252/T254/E256 (SEQ ID NO:52):

APEFLGGPSV FLFPPKPKDT L Y I T R E PEVT CVVVDVSQEDPEVQFNWYVD GVEVHNAKTK PREEQFNSTY RVVSVLTVLHQDWLNGKEYK CKVSNKGLPS SIEKTISKAK GQPREPQVYTLPPSQEEMTK NQVSLTCLVK GFYPSDIAVE WESNGQPENNYKTTPPVLDS DGSFFLYSRL TVDKSRWQEG NVFSCSVMHE ALHNHYTQKS LSLSLG X

-   -   wherein X is a lysine (K) or is absent.

A “knob-bearing” variant of such an IgG4 CH2-CH3 amino acid sequence hasthe amino acid sequence of SEQ ID NO:53:

APEFLGGPSV FLFPPKPKDT L Y I T R E PEVT CVVVDVSQEDPEVQFNWYVD GVEVHNAKTK PREEQFNSTY RVVSVLTVLHQDWLNGKEYK CKVSNKGLPS SIEKTISKAK GQPREPQVYT LPPSQEEMTK NQVSL WCLVK GFYPSDIAVE WESNGQPENN YKTTPPVLDS DGSFFLYSRL TVDKSRWQEG NVFSCSVMHEALHNHYTQKS LSLSLG X

-   -   wherein X is a lysine (K) or is absent.

A “hole-bearing” variant of such an IgG4 CG2-CH3 amino acid sequence hasthe amino acid sequence of SEQ ID NO:54:

APEFLGGPSV FLFPPKPKDT L Y I T R E PEVT CVVVDVSQEDPEVQFNWYVD GVEVHNAKTK PREEQFNSTY RVVSVLTVLHQDWLNGKEYK CKVSNKGLPS SIEKTISKAK GQPREPQVYT LPPSQEEMTK NQVSL S CA VK GFYPSDIAVE WESNGQPENN YKTTPPVLDS DGSFFL V SRL TVDKSRWQEG NVFSCSVMHEALHN R YTQKS LSLSLG X

-   -   wherein X is a lysine (K) or is absent.

It is preferred that the first polypeptide chain will have a“knob-bearing” CH2-CH3 sequence, such as that of SEQ ID NO:48 or SEQ IDNO:49. However, as will be recognized, a “hole-bearing” CH2-CH3 Domain(e.g., SEQ ID NO:50 or SEQ ID NO:51) could be employed in the firstpolypeptide chain, in which case, a “knob-bearing” CH2-CH3 Domain (e.g.,SEQ ID NO:48 or SEQ ID NO:49) would be employed in the secondpolypeptide chain of an Fc Domain-containing molecule of the presentinvention having two polypeptide chains (or in the third polypeptidechain of an Fc Domain-containing molecule having three, four, or fivepolypeptide chains).

In other embodiments, the invention encompasses Fc Domain-containingBinding Molecules comprising CH2 and/or CH3 Domains that have beenengineered to favor heterodimerization over homodimerization usingmutations known in the art, such as those disclosed in PCT PublicationNo. WO 2007/110205; WO 2011/143545; WO 2012/058768; WO 2013/06867, allof which are incorporated herein by reference in their entirety.

III. Trivalent Binding Molecules Containing Fc Domains

A further embodiment of the present invention relates to trivalentBinding Molecules comprising an Fc Domain capable of simultaneouslybinding a first epitope, a second epitope and a third epitope, whereinat least one of such epitopes is not identical to another. Suchtrivalent Binding Molecules comprise three Epitope-Binding Domains, twoof which are Diabody-Type Binding Domains, which provide binding Site Aand binding Site B, and one of which is a Fab-Type Binding Domain, or anscFv-Type Binding Domain, which provides binding Site C (see, e.g.,FIGS. 6A-6F, PCT Publication Nos. WO 2015/184207 and WO 2015/184203).Such trivalent Binding Molecules thus comprise “VL1”/“VH1” domains thatare capable of binding the first epitope and “VL2”/“VH2” domains thatare capable of binding the second epitope and “VL3” and “VH3” domainsthat are capable of binding the “third” epitope of such trivalentbinding molecule. A “Diabody-Type Binding Domain” is the type ofEpitope-Binding Domain present in a diabody, as described above. Each ofa “Fab-Type Binding Domain” and an “scFv-Type Binding Domain” areEpitope-Binding Domains that are formed by the interaction of the VLDomain of an immunoglobulin Light Chain and a complementing VH Domain ofan immunoglobulin Heavy Chain. Fab-Type Binding Domains differ fromDiabody-Type Binding Domains in that the two polypeptide chains thatform a Fab-Type Binding Domain comprise only a single Epitope-BindingDomain, whereas the two polypeptide chains that form a Diabody-TypeBinding Domain comprise at least two Epitope-Binding Domains. Similarly,scFv-Type Binding Domains also differ from Diabody-Type Binding Domainsin that they comprise only a single Epitope-Binding Domain. Thus, asused herein Fab-Type, and scFv-Type Binding Domains are distinct fromDiabody-Type Binding Domains.

Typically, the trivalent Binding Molecules of the present invention willcomprise four different polypeptide chains (see FIGS. 6A-6B), however,the molecules may comprise fewer or greater numbers of polypeptidechains, for example by fusing such polypeptide chains to one another(e.g., via a peptide bond) or by dividing such polypeptide chains toform additional polypeptide chains, or by associating fewer oradditional polypeptide chains via disulfide bonds. FIGS. 6C-6Fillustrate this aspect of the present invention by schematicallydepicting such molecules having three polypeptide chains. As provided inFIGS. 6A-6F, the trivalent Binding Molecules of the present inventionmay have alternative orientations in which the Diabody-Type BindingDomains are N-terminal (FIGS. 6A, 6C and 6D) or C-terminal (FIGS. 6B, 6Eand 6F) to an Fc Domain. CH2 and CH3 Domains useful for the generationof trivalent Binding Molecules are provided above and includeknob-bearing and hole-bearing domains.

In certain embodiments, the first polypeptide chain of such trivalentBinding Molecules of the present invention contains: (i) aVL1-containing Domain, (ii) a VH2-containing Domain, (iii) aHeterodimer-Promoting Domain, and (iv) a Domain containing a CH2-CH3sequence. The VL1 and VL2 Domains are located N-terminal or C-terminalto the CH2-CH3-containing domain as presented in Table 4 (also see,FIGS. 6A and 6B). The second polypeptide chain of such embodimentscontains: (i) a VL2-containing Domain, (ii) a VH1-containing Domain, and(iii) a Heterodimer-Promoting Domain. The third polypeptide chain ofsuch embodiments contains: (i) a VH3-containing Domain, (ii) aCH1-containing Domain and (iii) a Domain containing a CH2-CH3 sequence.The third polypeptide chain may be the Heavy Chain of an antibody thatcontains a VH3 and a Heavy Chain constant region, or a polypeptide thatcontains such domains. The fourth polypeptide of such embodimentscontains: (i) a VL3-containing Domain and (ii) a CL-containing Domain.The fourth polypeptide chains may be a Light Chain of an antibody thatcontains a VL3 complementary to the VH3 of the third polypeptide chain,or a polypeptide that contains such domains. The third or fourthpolypeptide chains may be isolated from naturally occurring antibodies.Alternatively, they may be constructed recombinantly, synthetically orby other means.

The Light Chain Variable Domain of the first and second polypeptidechains are separated from the Heavy Chain Variable Domains of suchpolypeptide chains by an intervening spacer peptide having a length thatis too short to permit their VL1/VH2 (or their VL2/VH1) Domains toassociate together to form Epitope-Binding Domain capable of bindingeither the first or second epitope. A preferred intervening spacerpeptide (Linker 1) for this purpose has the sequence (SEQ ID NO:16):GGGSGGGG. Other Domains of the trivalent Binding Molecules may beseparated by one or more intervening spacer peptides (Linkers),optionally comprising a cysteine residue. In particular, as providedabove, such Linkers will typically be incorporated between VariableDomains (i.e., VH or VL) and peptide Heterodimer-Promoting Domains(e.g., an E-coil or K-coil) and between such peptideHeterodimer-Promoting Domains (e.g., an E-coil or K-coil) and CH2-CH3Domains. Exemplary Linkers useful for the generation of trivalentBinding Molecules are provided above and are also provided in PCTApplication Nos: PCT/US15/33081; and PCT/US15/33076. Thus, the first andsecond polypeptide chains of such trivalent Binding Molecules associatetogether to form a VL1/VH1 binding site capable of binding a firstepitope, as well as a VL2/VH2 binding site that is capable of binding asecond epitope. The third and fourth polypeptide chains of suchtrivalent Binding Molecules associate together to form a VL3/VH3 bindingsite that is capable of binding a third epitope.

As described above, the trivalent Binding Molecules of the presentinvention may comprise three polypeptides. Trivalent Binding Moleculescomprising three polypeptide chains may be obtained by linking thedomains of the fourth polypeptide N-terminal to the VH3-containingDomain of the third polypeptide (e.g., using an intervening spacerpeptide (Linker 4)). Alternatively, a third polypeptide chain of atrivalent binding molecule of the invention containing the followingdomains is utilized: (i) a VL3-containing Domain, (ii) a VH3-containingDomain, and (iii) a Domain containing a CH2-CH3 sequence, wherein theVL3 and VH3 are spaced apart from one another by an intervening spacerpeptide that is sufficiently long (at least 9 or more amino acidresidues) so as to allow the association of these domains to form anEpitope-Binding Domain. One preferred intervening spacer peptide forthis purpose has the sequence: GGGGSGGGGSGGGGS (SEQ ID NO:41).

It will be understood that the VL1/VH1, VL2/VH2, and VL3/VH3 Domains ofsuch trivalent Binding Molecules may be different so as to permitbinding that is monospecific, bispecific or trispecific. In particular,the VL and VH Domains may be selected such that a trivalent bindingmolecule comprises two binding sites for a first epitope and one bindingsites for a second epitope, or one binding site for a first epitope andtwo binding sites for a second epitope, or one binding site for a firstepitope, one binding site for a second epitope and one binding site fora third epitope.

The general structure of the polypeptide chains of representativetrivalent Binding Molecules of invention is provided in FIGS. 6A-6F andin Table 5:

TABLE 5 Four 2^(nd )Chain NH₂—VL2—VH1—HPD—COOH Chain 1^(st )ChainNH₂—VL1—VH2—HPD—CH2—CH3—COOH 1^(st )Orien- 3^(rd )ChainNH₂—VH3—CH1—CH2—CH3—COOH tation 2^(nd )Chain NH₂—VL3—CL—COOH Four2^(nd )Chain NH₂—VL2—VH1—HPD—COOH Chain 1^(st )ChainNH₂—CH2—CH3—VL1—VH2—HPD—COOH 2nd Orien- 3^(rd )ChainNH₂—VH3—CH1—CH2—CH3—COOH tation 2^(nd )Chain NH₂—VL3—CL—COOH Three2^(nd )Chain NH₂—VL2—VH1—HPD—COOH Chain 1^(st )ChainNH₂—VL1—VH2—HPD—CH2—CH3—COOH 1st Orien- 3^(rd )ChainNH₂—VL3—VH3—HPD—CH2—CH3—COOH tation Three 2^(nd )ChainNH₂—VL2—VH1—HPD—COOH Chain 1^(st )Chain NH₂—CH2—CH3—VL1—VH2—HPD—COOH2^(nd )Orien- 3^(rd )Chain NH₂—VL3—VH3—HPD—CH2—CH3—COOH tation HPD= Heterodimer-Promoting Domain

As provided above, such trivalent Binding Molecules may comprise three,four, five, or more polypeptide chains.

IV. Embodiments of the Invention

As stated above, the present invention is directed to DA×CD3 BindingMolecules comprising a vCD3-Binding Domain that comprises a CDR_(H)1Domain, a CDR_(H)2 Domain, a CDR_(H)3 Domain, a CDR_(L)1 Domain, aCDR_(L)2 Domain, and a CDR_(L)3 Domain, at least one of which differs inamino acid sequence from the amino acid sequence of the correspondingCDR of an rCD3-Binding Domain. The rCD3-Binding Domain that is to beemployed in such comparison with a particular vCD3-Binding Domain is theCD3-Binding Domain of an isolated CD3-binding antibody that exhibits thegreatest identity of CDR sequence with such particular vCD3-BindingDomain. The rCD3-Binding Domain preferably also exhibits at least 95% to100% identity in the framework regions. A preferred rCD3-Binding Domaincomprises the CDR_(H)1 Domain, CDR_(H)2 Domain, CDR_(H)3 Domain,CDR_(L)1 Domain, CDR_(L)2 Domain, and CDR_(L)3 Domain of CD3 mAb-1. TheDA×CD3 Binding Molecules of the present invention that comprise suchvCD3-Binding Domain exhibit an altered affinity for CD3, relative to aDA×CD3 Binding Molecule comprising such rCD3-Binding Domain. Theinvention particularly concerns to such DA×CD3 Binding Moleculescomprising a vCD3-Binding Domain which exhibit reduced affinity for CD3and are capable of mediating redirected killing of target cellsexpressing a Disease Antigen, and exhibit reduced levels of cytokinerelease relative to a DA×CD3 Binding Molecule comprising a rCD3-BindingDomain. The invention particularly concerns the use of DA×CD3 BindingMolecules comprising a vCD3-Binding Domain in the treatment of cancerand pathogen-associated diseases. The present invention is also directedto pharmaceutical compositions that comprise such molecule(s).

The invention thus encompasses DA×CD3 Binding Molecules comprising oneor more of the VH and/or VL Domains of a vCD3-Binding Domain, or morepreferably, the CDR_(H)1, CDR_(H)2, and CDR_(H)3, and the CDR_(L)1,CDR_(L)2 and CDR_(L)3 portions of such Domains. In a preferredembodiment of the invention, such DA×CD3 Binding Molecules BindingMolecules will additionally contain binding domains sufficient to permitsuch molecules to bind to epitope(s) of one, two, or more DiseaseAntigens. In another preferred embodiment of the invention, such DA×CD3Binding Molecules will additional contain binding domains sufficient topermit such molecules to bind to epitope(s) of another moleculeexpressed on the surface of an effector cell, such as CD2, CD8, CD16,T-cell Receptor (TCR), NKp46, NKG2D, etc., which are expressed on Tlymphocytes, Natural Killer (NK) cells, Antigen-Presenting Cells orother mononuclear cells).

The present invention is also directed to pharmaceutical compositionsthat comprise such DA×CD3 Binding Molecule(s).

By possessing binding domains sufficient to immunospecifically bind CD3and a Disease Antigen, the molecules of the present invention have theability to mediate the redirected killing of a target cell (e.g., acancer cell or a pathogen-infected cell) that arrays the Disease Antigenon its surface. The combined presence of both such binding affinitiesserves to localize the a CD3-expressing effector cell to the site of thetarget cell (i.e., to “redirect” the effector cell) so that it maymediate the killing of the target cell. As discussed above, suchmolecules may be bispecific, or may be capable of binding more than twoepitopes (e.g., trispecific).

Efforts to employ CD3 Binding Molecules have been encumbered by the highmagnitude of immune activation caused by such therapies and theattendant and adverse production of high levels of cytokines in somepatients. Thus, although anti-CD3 therapies have resulted in asignificant degree of immune activation in recipient patients, which hascorrelated with greatly increased efficacy, the use of such moleculeshas been associated with notable toxicity (Frey, N. V. et al. (2016)“Cytokine Release Syndrome With Novel Therapeutics For AcuteLymphoblastic Leukemia,” Hematol. Am. Soc. Hematol. Educ Program.(1):567-572; Teachey, D. T. et al. (2013) “Cytokine Release SyndromeAfter Blinatumomab Treatment Related To Abnormal Macrophage ActivationAnd Ameliorated With Cytokine-Directed Therapy,” Blood121(26):5154-5157; Le Jeune, C. et al. (2016) “Potential For BispecificT-Cell Engagers: Role Of Blinatumomab In Acute Lymphoblastic Leukemia,”Drug Des. Devel. Ther. 10:757-765; Newman, M. J. et al. (2016) “A ReviewOf Blinatumomab, A Novel Immunotherapy,” J. Oncol. Pharm. Pract.22(4):639-645; Fitzgerald, J. C. et al. (2017) “Cytokine ReleaseSyndrome After Chimeric Antigen Receptor T-Cell Therapy for AcuteLymphoblastic Leukemia,” Crit. Care Med. 45(2):e124-e131; Teachey, D. T.et al. (2016) “Identification of Predictive Biomarkers for CytokineRelease Syndrome after Chimeric Antigen Receptor T-cell Therapy forAcute Lymphoblastic Leukemia,” Cancer Discov. 6(6):664-679; Goebeler, M.E. et al. (2016) “Blinatumomab: A CD19/CD3 Bispecific T Cell Engager(Bite) With Unique Anti-Tumor Efficacy,” Leuk. Lymphoma 57(5):1021-1032;Barrett, D. M. et al. (2014) “Toxicity Management For Patients ReceivingNovel T-Cell Engaging Therapies,” Curr. Opin. Pediatr. 26(1):43-49).

The present invention addresses such encumbrance by demonstrating thatparental CD3-Binding Domains (i.e., rCD3-Binding Domains) that exhibitboth high cytotoxicity and high cytokine release when incorporated intoDA×CD3 Binding Molecules may be engineered to produce variants (i.e.,vCD3-Binding Domains) having altered affinity for CD3 that are capableof mediating redirected killing and exhibit reduced levels of cytokinerelease relative to a DA×CD3 Binding Molecule comprising a rCD3-BindingDomain. In particular, DA×CD3 Binding Molecules comprising avCD3-Binding Domains of the invention exhibit reduced levels of releaseof any one or more of: IFN-γ, TNF-α, IL-2, and/or IL-6.

The present invention stems, in part, from the recognition thatcytotoxicity and cytokine release are separable properties of DA×CD3Binding Molecules. The present invention encompasses variant CD3-BindingDomains (i.e., vCD3-Binding Domains) that retain high levels ofcytotoxicity while exhibiting reduced levels of cytokine release, andthe use of DA×CD3 Binding Molecules comprising such vCD3-Binding Domainsin the treatment of disease. As used herein, the term “variant” withrespect to such CD3-Binding Domains is intended to refer to CD3-BindingDomains having at least one CDR_(H), and/or at least one CDR_(L), thatdiffers from the “corresponding” CDR_(H) and/CDR_(L) of a “reference”CD3-Binding Domain (i.e., rCD3-Binding Domain). As used herein the term“corresponding” CDR_(H) and/CDR_(L) denotes a comparison between two CDRsequences in which both such CDRs are CD_(H)1 Domains, both such CDRsare CD_(H)2 Domains, both such CDRs are CD_(H)3 Domains, both such CDRsare CD_(L)1 Domains, both such CDRs are CD_(L)2 Domains, or both suchCDRs are CD_(L)3 Domains. A preferred rCD3-binding domain for theexemplary vCD3-binding domains described herein is a CD3-Binding Domainhaving at least 5, at least 4, at least 3, at least 2 or at least 1 ofthe CDRs: CD_(H)1, CDR_(H)2, CDR_(H)3 and CD_(L)1, CDR_(L)2, andCDR_(L)3 of CD3 mAb 1. Preferably, such exemplary vCD3-binding domainswill possess at least 5 of the CDRs: CD_(H)1, CDR_(H)2, CDR_(H)3 andCD_(L)1, CDR_(L)2, and CDR_(L)3 of CD3 mAb 1. vCD3-binding domains maybe obtained through the chemical modification of one or more CDRs of therCD3-Binding Domain, but will more preferably be obtained by forming oneor more polynucleotides that encode such one or more CDRs of therCD3-binding Domain, except being altered to encode the desiredvCD3-Binding Domain, and then expressing such polynucleotide in anappropriate protein expression system (e.g., a cell, or in vitrotranslation system). Cytotoxicity may be measured in any suitable manner(e.g., a CTL assay to determine the EC₅₀, maximum, etc.). Cytokinerelease may be measured by assaying for any one or more of: IFN-gamma,TNF-alpha, IL-6 or IL-2 in any suitable manner (e.g., a CTL assay todetermine the EC₅₀, maximum, etc.).

Notably, the absolute levels of maximal cytotoxicity and cytokinerelease are not the only criteria used to assess whether a candidateCD3-Binding Domain is a suitable vCD3-Binding Domain encompassed by thepresent invention. In addition, or alternatively, EC₅₀ values may beemployed. As provided herein, a suitable vCD3-Binding Domain is onethat, when incorporated into a DA×CD3 Binding Molecule, is capable ofmediating high levels of cytotoxicity (i.e., a low EC₅₀ concentration)while exhibiting reduced levels of cytokine release.

In certain embodiments, the instant invention provides a vCD3-BindingDomain that, when incorporated into a DA×CD3 Binding Molecule, mediatescell redirected cell killing to a maximum cytotoxicity (e.g., asmeasured in a CTL assay at 18-48 hours) that is at least about 60%, atleast about 70%, at least about 80%, at least about 90%, at least about100%, of that mediated by a DA×CD3 Binding Molecule comprising arCD3-Binding Domain. Additionally, or alternatively, a DA×CD3 BindingMolecule comprising a vCD3-Binding Domains of the invention exhibits anEC₅₀ of cytotoxicity (e.g., a measured in a CTL assay at 18-48 hours)that is increased by less than about 10%, less than about 20%, less thanabout 30%, less than about 40%, less than about 50%, less than about60%, less than about 70%, less than about 80%, less than about 90%, lessthan about 100%, less than about 200%, less than about 300%, less thanabout 400%, or less than about 500% of that exhibited by a DA×CD3Binding Molecule comprising a rCD3-Binding Domain. Additionally, oralternatively the ratio of the EC₅₀ of cytotoxicity (e.g., as measuredin a CTL assay at 18-24 hours) of a DA×CD3 Binding Molecule comprising avCD3-Binding Domain of the invention to a DA×CD3 Binding Moleculecomprising the rCD3-Binding Domain (EC₅₀ variant/EC₅₀ reference) is lessthan about 2, is less than about 5, is less than about 10, is less thanabout 20, is less than about 40, is less than about 60, is less thanabout 80, is less than about 100, or is less than about 200.

In certain embodiments, a DA×CD3 Binding Molecule comprising avCD3-Binding Domains of the invention exhibits a maximum release of oneor more cytokine (e.g., as measured in a CTL assay at 18-24 hours) thatis reduced by at least about 10%, at least about 20%, at least about30%, at least about 40%, at least about 50%, at least about 60%, atleast about 70%, at least about 80%, at least about 90%, or more of thatexhibited by a DA×CD3 Binding Molecule comprising a rCD3-Binding Domain.Additionally, or alternatively, DA×CD3 Binding Molecules comprising thevCD3-Binding Domains of the invention exhibit an EC₅₀ of release of oneor more cytokine (e.g., as measured in a CTL assay at 18-48 hours) thatis increased by at least about 10%, at least about 20%, at least about30%, at least about 40%, at least about 50%, at least about 60%, atleast about 70%, at least about 80, at least about 90%, or more of thatexhibited by a DA×CD3 Binding Molecule comprising a rCD3-Binding Domain.In particular embodiments, the cytokine released is selected from thegroup consisting of: IFN-γ, TNF-α, IL-2, and IL-6. Additionally, oralternatively the ratio of the EC₅₀ of release of one or more cytokine(e.g., as measured in a CTL assay at 18-24 hours) of a DA×CD3 BindingMolecule comprising a vCD3-Binding Domain of the invention to a DA×CD3Binding Molecule comprising the rCD3-Binding Domain (EC₅₀ variant/EC₅₀reference) is more that about 1, is more than about 2, is more thanabout 5, is more than about 10, is more than about 20, is more thanabout 40, is more than about 60, is more than about 80, is more thanabout 100, or is more than about 200.

Additionally, DA×CD3 Binding Molecules comprising a vCD3-Binding Domainof the invention retain at least about 50%, at least about 60%, at leastabout 70%, at least about 80%, at least about 90%, at least about 100%,of an in vivo activity (e.g., anti-tumor, anti-pathogen activity)exhibited by a DA×CD3 Binding Molecule comprising a rCD3-Binding Domain.In view of the instant disclosure it will be understood that DA×CD3Binding Molecules comprising a vCD3-Binding Domain may be administeredat a higher dose to achieve an in vivo activity that is at least about50% or more of that exhibited by a DA×CD3 Binding Molecule comprising arCD3-Binding Domain, but that such higher dose will exhibit reducedlevels of cytokine release as compared to the DA×CD3 Binding Moleculecomprising a rCD3-Binding Domain.

In one embodiment, such DA×CD3 Binding Molecules of the presentinvention will be monospecific so as to possess the ability to bind toonly a single epitope of CD3 and only a single epitope of the DiseaseAntigen.

Alternatively, such DA×CD3 Binding Molecules may be multispecific, i.e.,capable of binding 1, 2, 3, 4, or more than 4 epitopes, which may beapportioned in any manner to bind 1, 2, or more epitope(s) of CD3 and 1,2, 3, 4, or more than 4 epitope(s) of one or more Disease Antigen(s).

In certain embodiments, where such DA×CD3 Binding Molecules are capableof immunospecifically binding to only a single Disease Antigen, they maybe capable of immunospecifically binding to only one CD3 epitope and toone, two epitope(s) of such Disease Antigen (which two Disease Antigenepitopes may be the same or different), or they may be capable ofimmunospecifically binding to only one CD3 epitope and to threeepitope(s) of such Disease Antigen (which three Disease Antigen epitopesmay be the same, or may be different, or may be two epitopes that arethe same and one epitope that is different).

In other embodiments, where such DA×CD3 Binding Molecules are capable ofimmunospecifically binding to two different Disease Antigens (e.g., aFirst Disease Antigen and a Second Disease Antigen), they may be capableof immunospecifically binding to only one CD3 epitope and to one or twoepitope(s) of the First Disease Antigen (which two First Disease Antigenepitopes may be the same or different) and two or one epitope(s) of theSecond Disease Antigen (which two Second Disease Antigen epitopes may bethe same or different).

In still other embodiments, such DA×CD3 Binding Molecules may be capableof immunospecifically binding to three different Disease Antigens (e.g.,a First Disease Antigen, a Second Disease Antigen and a Third DiseaseAntigen) and only one CD3 epitope.

In still other embodiments, such DA×CD3 Binding Molecules may be capableof immunospecifically binding to one or two different Disease Antigens(e.g., a First Disease Antigen and a Second Disease Antigen), only oneCD3 epitope, and one or two different cell surface molecules (which maybe the same cell surface molecule or may be different surface molecules)of an effector cell (which may be the same type of effector cell or maybe a different type of effector cell).

Thus, for example, such DA×CD3 Binding Molecules may bind:

-   -   (1) a single epitope of CD3 and a single epitope of a Disease        Antigen that is arrayed on the surface of the target cell;    -   (2) a single epitope of CD3 and two epitopes of the same Disease        Antigen that is arrayed on the surface of the target cell;    -   (3) a single epitope of CD3, an epitope of a First Disease        Antigen that is arrayed on the surface of the target cell and an        epitope of a Second Disease Antigen that is arrayed on the        surface of the target cell;    -   (4) a single epitope of CD3 and three epitopes of the same        Disease Antigen that is arrayed on the surface of the target        cell;    -   (5) a single epitope of CD3, two epitopes of a First Disease        Antigen that is arrayed on the surface of the target cell, and        one epitope of a Second Disease Antigen that is arrayed on the        surface of the target cell;    -   (6) a single epitope of CD3, an epitope of a First Disease        Antigen that is arrayed on the surface of the target cell, and        an epitope of a Second Disease Antigen that is arrayed on the        surface of the target cell;    -   (7) a single epitope of CD3, a single epitope of a Disease        Antigen that is arrayed on the surface of the target cell and a        single epitope of a cell surface molecule other than CD3 that is        arrayed on the surface of an effector cell (which may be the        same type of effector cell as that arraying CD3 or may be a        different type of effector cell);    -   (8) a single epitope of CD3, two epitopes of a Disease Antigen        that is arrayed on the surface of the target cell and a single        epitope of a cell surface molecule other than CD3 that is        arrayed on the surface of an effector cell (which may be the        same type of effector cell as that arraying CD3 or may be a        different type of effector cell);    -   (9) a single epitope of CD3, an epitope of a First Disease        Antigen that is arrayed on the surface of the target cell, an        epitope of a Second Disease Antigen that is arrayed on the        surface of the target cell and a single epitope of a cell        surface molecule other than CD3 that is arrayed on the surface        of an effector cell (which may be the same type of effector cell        as that arraying CD3 or may be a different type of effector        cell);    -   (10) a single epitope of CD3, an epitope of a Disease Antigen        that is arrayed on the surface of the target cell, and two        epitopes of a cell surface molecule other than CD3 that is        arrayed on the surface of an effector cell (which may be the        same type of effector cell as that arraying CD3 or may be a        different type of effector cell); or    -   (11) a single epitope of CD3, an epitope of a Disease Antigen        that is arrayed on the surface of the target cell, an epitope of        a first cell surface molecule other than CD3 that is arrayed on        the surface of an effector cell (which may be the same type of        effector cell as that arraying CD3 or may be a different type of        effector cell), and an epitope of a second cell surface molecule        other than CD3 that is arrayed on the surface of an effector        cell (which may be the same type of effector cell as that        arraying CD3 or may be a different type of effector cell).

The invention thus contemplates DA×CD3 Binding Molecules that comprise afirst Epitope-Binding Domain capable of immunospecifically binding anepitope of CD3 and a second Epitope-Binding Domain that is capable ofimmunospecifically binding an epitope of a Disease Antigen that isarrayed on the surface of such target cell and a third Epitope-BindingDomain capable of immuno specifically binding an epitope of a differentcell surface molecule of an effector cell (which may be the same type ofeffector cell or may be a different type of effector cell). In aspecific embodiment, the different cell surface molecule of an effectorcell is CD8. Table 6 illustrates possible combination bindingspecificities of exemplary molecules of the invention.

TABLE 6 Number of Epitopes Recognized by Exemplary Molecules of theInvention Capable of Mediating the Redirected Killing of a Target CellTotal Other 1^(st) 2^(nd) 3^(nd) Number of Effector Disease DiseaseDisease Binding CD3 Cell Antigen Antigen Antigen Domains Epitope EpitopeEpitope Epitope Epitope 2 1 0 1 0 0 3 1 0 1 1 0 3 1 1 1 0 0 3 1 0 1 1 03 2 0 1 0 0 4 1 0 1 1 1 4 1 0 1 2 0 4 1 0 2 1 0 4 1 1 2 0 0 4 1 1 1 1 04 1 2 1 0 0 4 2 0 1 1 0 4 2 0 1 1 0 4 2 1 1 0 0

By forming more complex molecules, one may obtain DA×CD3 BindingMolecules that are capable of binding CD3 and one or more DiseaseAntigens and optionally a different cell surface molecule of an effectorcell that possess more than four Epitope-Binding Domains. No limitationis placed on the nature or number of epitopes or additional epitopesthat may be bound by the molecules of the present invention other thanthat such additional binding capability does not prevent the molecule orBinding Domain thereof that is capable of binding to an epitope of CD3from such binding and does not prevent the molecule or Binding Domainthereof that is capable of binding to an epitope of a Disease Antigenfrom such binding, so that the molecule(s) may mediate the redirectedkilling of the target cell.

V. Exemplary Binding Molecules

The present invention is directed to DA×CD3 Binding Molecules (e.g., adiabody, a bispecific antibody, a bispecific, a trivalent molecule, aBiTe, a TandAb, etc.) capable of binding to CD3 and a Disease Antigen,such as a Cancer Antigen or a Pathogen-Associated Antigen. Such BindingMolecules can be readily produced from the CDRs of antibodies and fromthe VL and VH Domains of antibodies. Listed below are exemplaryantibodies that may be used to produce the Binding Molecules andcombination therapy of the present invention.

A. Anti-CD3 Antibody CD3 mAb 1

The present invention employs variant CD3-Binding Domains (i.e.,vCD3-Binding Domains) that comprise the Light Chain Variable (VL) Domainand the Heavy Chain Variable (VH) Domain of anti-human CD3 antibodies,or CD3-binding portions thereof, and that mediate variant binding toCD3. As used herein, the term “variant binding” is intended to refer tothe comparative binding exhibited by the CD3-Binding Domains of areference antibody whose CDRs exhibit the highest sequence identity tothe CDRs of the variant CD3-Binding Domain. The CD3-binding referenceantibody for the illustrative vCD3-Binding Domains of the presentinvention is CD3 mAb 1, whose rCD3-Binding Domain is capable of bindinghuman CD3 and CD3 of non-human primates (e.g., cynomolgus monkey).

The amino acid sequence of the VH Domain of CD3 mAb 1 (SEQ ID NO:55) isshown below (CDR_(H) residues are shown underlined):

EVQLVESGGG LVQPGGSLRL SCAASGFTFS  TYAMN WVRQA PGKGLEWVG R  IRSKYNNYAT YYADSVK  

RF TISRDDSKNS LYLQMNSLKT EDTAVYYCVR  HGNFGNSYVS WFAY WGQGTL VTVSS

-   -   wherein X is aspartate (D) or glycine (G)

The amino acid sequence of the VL Domain of CD3 mAb 1 (SEQ ID NO:56) isshown below (CDR_(L) residues are shown underlined):

QAVVTQEPSL TVSPGGTVTL TC RSSTGAVT TSNYAN WVQQ KPGQAPRGLI G GTNKRAPWT PARFSGSLLG GKAALTITGA QAEDEADYYC  ALWYSNLWV F GGGTKLTVLG

CD3 mAb 1 CDR Sequence SEQ ID NO CDR_(H)1 TYAMN SEQ ID NO: 57 CDR_(H)2RIRSKYNNYATYYADSVK X SEQ ID NO: 58 CDR_(H)3 HGNFGNSYVSWFAY SEQ ID NO: 59CDR_(L)1 RSSTGAVTTSNYAN SEQ ID NO: 60 CDR_(L)2 GTNKRAP SEQ ID NO: 61CDR_(L)3 ALWYSNLWV SEQ ID NO: 62 wherein X is aspartate (D) or glycine(G)

The rCD3-Binding Domain of “CD3 mAb 1” comprises a CD3 mAb 1 VH Domainhaving either aspartate (D) or glycine (G) at Kabat position 65,corresponding to residue 68 of SEQ ID NO:55) (i.e., X in SEQ ID NO:55 isaspartate (D) or glycine (G)) and the VL Domain of CD3 mAb 1 (SEQ IDNO:56). Thus, for example, when such CD3 mAb 1 VH Domain has a glycine(G) as its residue 68, its sequence is SEQ ID NO:63, shown below(CDR_(H) residues are shown underlined, Kabat position 65 is shown indouble underline):

EVQLVESGGG LVQPGGSLRL SCAASGFTFS  TYAMN WVRQA PGKGLEWVG R  IRSKYNNYAT YYADSVK  

RF TISRDDSKNS LYLQMNSLKT EDTAVYYCVR  HGNFGNSYVS WFAY WGQGTL VTVSS

CD3-Binding Molecules that possess a vCD3-Binding Domain of the presentinvention may be recognized using a CTL assay in which:

-   -   (1) a bispecific Cancer Antigen×CD3 diabody (for example, a        CD123×CD3 diabody or a 5T4×CD3 diabody) potentially having a        vCD3-Binding Domain, and    -   (2) a bispecific Cancer Antigen×CD3 diabody having a        corresponding rCD3-Binding Domain (e.g., the rCD3-Binding Domain        of CD3 mAb 1),        are separately incubated with effector Pan-T-cells (or PBMCs)        and target tumor cells (e.g., MOLM-13 or A498 cells), for        example, at an effector:target cell ratio of 5:1 (or 15:1 for        PBMCs) for 18, 24, or 42 hours, and the percentage cytotoxicity        (i.e., cell killing) and/or EC₅₀ is determined (for example, by        measuring the release of lactate dehydrogenase (LDH) into the        media by damaged cells using the CytoTox 96® Non-Radioactive        Cytotoxicity Assay Kit (Promega)). In one embodiment, the        release of IFN-γ, TNF-α, IL-6, and IL-2 cytokines may be        determined at the end of the CTL assay. CD4+ and CD8+T        lymphocyte populations may also be assessed for up-regulation of        the activation markers CD69 and CD25 at the end of the CTL        assay. A comparison of the percentage cytotoxicity and/or EC₅₀        for the bispecific Cancer Antigen×CD3 diabody potentially having        a vCD3-Binding Domain with that of the Cancer Antigen×CD3        diabody having the rCD3-Binding Domains identifies vCD3-binding        domains that exhibit the desired variant CD3 binding and/or        reduced level of cytokine release.

CD3-Binding Molecules that possess a vCD3-Binding Domain of the presentinvention may alternatively be recognized using a binding assay inwhich:

-   -   (1) a bispecific Cancer Antigen×CD3 diabody potentially having a        vCD3-Binding Domain, and    -   (2) a bispecific Cancer Antigen×CD3 diabody having an        rCD3-Binding Domain (e.g., the rCD3-Binding Domain of CD3 mAb        1),        are separately evaluated for their ability to bind to the        surface of cells of tumor antigen-expressing cell lines (MOLM-13        or A498 cells) by FACS analysis. Briefly, cells are incubated        with the diabody molecules (in FACS buffer containing 10% human        AB serum) in microtiter plates. The cells are then washed and        incubated with a labeled anti-human Fc secondary antibody or        with a biotin-conjugated mouse anti-EK-coil antibody that        recognizes the E-coil/K-coil (EK) Heterodimer-Promoting Domain        of the diabodies, mixed with streptavidin-phycoerythrin. The        cells are then washed and resuspended with FACS buffer and        analyzed by flow cytometry and compared.

CD3-Binding Molecules that possess a vCD3-Binding Domain of the presentinvention may alternatively be recognized using, for example, a Co-MixXenograft Model such as NOD/SCID mice. In such an assay, the mice areinjected with tumor cells (e.g., KG1A (AML) cells) co-mixed withactivated human CD4+ or CD8+ T-cells (E:T=1:5). The bispecific CancerAntigen×CD3 diabody potentially having a vCD3-Binding Domain or theCancer Antigen×CD3 diabody having the rCD3-Binding Domain is injectedinto the animals and the extent of tumor growth is monitored andcompared.

Alternatively, any one, two, or more than two of the exemplary variantsof CD3 mAb 1, designated herein as “CD3 mAb 1 M3”-“CD3 mAb 1 M26” may beemployed to provide the vCD3-Binding Domain of the DA×CD3 BindingMolecules of the present invention. The invention fully contemplatesanti-CD3 antibodies having the VL and VH Domains of ant of CD3 mAb 1M3-CD3 mAb 1 M26 wherein the VH Domain possesses an aspartate (D) atKabat position 65 or a glycine (G) at Kabat position 65. The exemplaryvariants of CD3 mAb 1, CD3 mAb 1 M3-CD3 mAb 1 M26 possess vCD3-BindingDomains that comprise a CDR_(H)1 Domain, a CDR_(H)2 Domain, a CDR_(H)3Domain, a CDR_(L)1 Domain, a CDR_(L)2 Domain, and a CDR_(L)3 Domain, atleast one of which differs in amino acid sequence from the amino acidsequence of the corresponding CDR of the rCD3-Binding Domain (CD3 mAb1); and relative to a DA×CD3 Binding Domain comprising said rCD3-BindingDomain. a DA×CD3 Binding Molecule comprising said vCD3-Binding Domainbinds CD3 with an altered affinity and is capable of mediatingredirected killing and exhibit lower levels of cytokine release.

The amino acid sequences of preferred variant anti-CD3 VH Domains of thepresent invention are variants of SEQ ID NO:55 and are represented bySEQ ID NO:207 (CDR_(H) residues are shown underlined):

EVQLVESGGG LVQPGGSLRL SCAASGFTFS  X ₁ X ₂ X ₃ MN WVRQA PGKGLEWVGR IRSKYNNYAT   YYADSVKX ₄ RF TISRDDSKNS LYLQMNSLKT EDTAVYYCVR  HX ₅ NX ₆ X₇ NSX ₈ ST   X ₉ FAX ₁₀ WGQGTL VTVSSwherein: X₁ is T, D, or E; X₂ is Y, D or T; X₃ is A or G; X₄ is D or G;X₅ is G, D, E, or K; X₆ is F or I; X₇ is G or I; X₈ is Y, A, G, or Q; X₉is W, F, or Y; and X₁₀ is Y or E.

The amino acid sequences of preferred variant anti-CD3 VL Domains of thepresent invention are variants of SEQ ID NO:56 and are represented bySEQ ID NO:208 (CDR_(L) residues are shown underlined):

QAVVTQEPSL TVSPGGTVTL TC RSSTGAVT   TSNYAN WVQQ KPGQAPRGLI G X ₁ TNX ₂RAP WT PARFSGSLLG GKAALTITGA QAEDEADYYC  AX ₃ WYSNLWV F GGGTKLTVLGwherein: X₁ is G or D; X₂ is K or G; and X₃ is L, E or Q.

B. Variant Anti-CD3 Antibodies

1. CD3 mAb 1 M1

CD3 mAb 1 M1 is a low affinity variant of CD3 mAb 1, and is thus alsoreferred to as “CD3 mAb 1 Low.” The amino acid sequence of the VH Domainof CD3 mAb 1 M1 is shown below as SEQ ID NO:64 (CDR_(H) residues areshown underlined). Relative to SEQ ID NO:55, SEQ ID NO:64 contains anS100dT substitution (shown in double underline, and numbered as inKabat); additionally, position 65, numbered as in Kabat, of SEQ IDNO:64, also shown in double underline, may be aspartate (D) or glycine(G):

EVQLVESGGG LVQPGGSLRL SCAASGFTFS  TYAMN WVRQA PGKGLEWVG R  IRSKYNNYAT YYADSVK  

RF TISRDDSKNS LYLQMNSLKT EDTAVYYCVR  HGNFGNSYV  

 WFA YWGQGTL VTVSS

-   -   wherein X is aspartate (D) or glycine (G)

A preferred amino acid sequence of the VL Domain of CD3 mAb 1 M1 is SEQID NO:56.

CD3 mAb 1 M1 CDR Sequence SEQ ID NO CDR_(H)1 TYAMN SEQ ID NO: 57CDR_(H)2 RIRSKYNNYATYYADSVK X SEQ ID NO: 58 CDR_(H)3 HGNFGNSYV 

WFAY SEQ ID NO: 65 CDR_(L)1 RSSTGAVTTSNYAN SEQ ID NO: 60 CDR_(L)2GTNKRAP SEQ ID NO: 61 CDR_(L)3 ALWYSNLWV SEQ ID NO: 62

2. CD3 mAb 1 M2

CD3 mAb 1 M2 has a faster off-rate than CD3 mAb 1, and is thus alsoreferred to as “CD3 mAb 1 Fast.” The amino acid sequence of the VHDomain of CD3 mAb 1 M2 is shown below as SEQ ID NO:66 (CDR_(H) residuesare shown underlined). Relative to SEQ ID NO:55, SEQ ID NO:66 containsG96K and S100dT substitutions, numbered as in Kabat (sequence residue110, shown in double underline); additionally, position 65, numbered asin Kabat, of SEQ ID NO:66, also shown in double underline, may beaspartate (D) or glycine (G):

EVQLVESGGG LVQPGGSLRL SCAASGFTFS  TYAMN WVRQA PGKGLEWVG R  IRSKYNNYAT YYADSVK  

RF TISRDDSKNS LYLQMNSLKT EDTAVYYCVR  H  

NFGNSYV  

 WFAY WGQGTL VTVSS

-   -   wherein X is aspartate (D) or glycine (G)

A preferred amino acid sequence of the VL Domain of CD3 mAb 1 M2 is SEQID NO:56.

CD3 mAb 1 M2 CDR Sequence SEQ ID NO CDR_(H)1 TYAMN SEQ ID NO: 57CDR_(H)2 RIRSKYNNYATYYADSVK X SEQ ID NO: 58 CDR_(H)3 H 

NFGNSYV 

WFAY SEQ ID NO: 67 CDR_(L)1 RSSTGAVTTSNYAN SEQ ID NO: 60 CDR_(L)2GTNKRAP SEQ ID NO: 61 CDR_(L)3 ALWYSNLWV SEQ ID NO: 62 wherein X isaspartate (D) or glycine (G)

3. CD3 mAb 1 M3

The amino acid sequence of the VH Domain of CD3 mAb 1 M3 (SEQ ID NO:68)is shown below (CDR_(H) residues are shown underlined). Relative to SEQID NO:55, SEQ ID NO:68 contains a G999 substitution (shown in doubleunderline, and numbered as in Kabat); additionally, position 65,numbered as in Kabat, of SEQ ID NO:68, also shown in double underline,may be aspartate (D) or glycine (G):

EVQLVESGGG LVQPGGSLRL SCAASGFTFS  TYAMN WVRQA PGKGLEWVG R  IRSKYNNYAT YYADSVK  

RF TISRDDSKNS LYLQMNSLKT EDTAVYYCVR  HGNF  

NSYVS WFAY WGQGTL VTVSS

-   -   wherein X is aspartate (D) or glycine (G)

A preferred amino acid sequence of the VL Domain of CD3 mAb 1 M3 is SEQID NO:56.

CD3 mAb 1 M3 CDR Sequence SEQ ID NO CDR_(H)1 TYAMN SEQ ID NO: 57CDR_(H)2 RIRSKYNNYATYYADSVK X SEQ ID NO: 58 CDR_(H)3 HGNF 

NSYVSWFAY SEQ ID NO: 69 CDR_(L)1 RSSTGAVTTSNYAN SEQ ID NO: 60 CDR_(L)2GTNKRAP SEQ ID NO: 61 CDR_(L)3 ALWYSNLWV SEQ ID NO: 62 wherein X isaspartate (D) or glycine (G)

4. CD3 mAb 1 M4

The amino acid sequence of the VH Domain of CD3 mAb 1 M4 (SEQ ID NO:70)is shown below (CDR_(H) residues are shown underlined). Relative to SEQID NO:55, SEQ ID NO:70 contains a Y100bA substitution (shown in doubleunderline, and numbered as in Kabat); additionally, position 65,numbered as in Kabat, of SEQ ID NO:70, also shown in double underline,may be aspartate (D) or glycine (G):

EVQLVESGGG LVQPGGSLRL SCAASGFTFS  TYAMN WVRQA PGKGLEWVG R  IRSKYNNYAT YYADSVK  

RF TISRDDSKNS LYLQMNSLKT EDTAVYYCVR  HGNFGNS  

VS WFAY WGQGTL VTVSS

-   -   wherein X is aspartate (D) or glycine (G)

A preferred amino acid sequence of the VL Domain of CD3 mAb 1 M4 is SEQID NO:56.

CD3 mAb 1 M4 CDR Sequence SEQ ID NO CDR_(H)1 TYAMN SEQ ID NO: 57CDR_(H)2 RIRSKYNNYATYYADSVK X SEQ ID NO: 58 CDR_(H)3 HGNFGNS 

VSWFAY SEQ ID NO: 71 CDR_(L)1 RSSTGAVTTSNYAN SEQ ID NO: 60 CDR_(L)2GTNKRAP SEQ ID NO: 61 CDR_(L)3 ALWYSNLWV SEQ ID NO: 62 wherein X isaspartate (D) or glycine (G)

5. CD3 mAb 1 M5

The amino acid sequence of the VH Domain of CD3 mAb 1 M5 (SEQ ID NO:72)is shown below (CDR_(H) residues are shown underlined). Relative to SEQID NO:55, SEQ ID NO:72 contains a Y100bG substitution (shown in doubleunderline, and numbered as in Kabat); additionally, position 65,numbered as in Kabat, of SEQ ID NO:72, also shown in double underline,may be aspartate (D) or glycine (G):

EVQLVESGGG LVQPGGSLRL SCAASGFTFS  TYAMN WVRQA PGKGLEWVG R  IRSKYNNYAT YYADSVK  

RF TISRDDSKNS LYLQMNSLKT EDTAVYYCVR  HGNFGNS  

VS WFAY WGQGTL VTVSS

-   -   wherein X is aspartate (D) or glycine (G)

A preferred amino acid sequence of the VL Domain of CD3 mAb 1 M5 is SEQID NO:56.

CD3 mAb 1 M5 CDR Sequence SEQ ID NO CDR_(H)1 TYAMN SEQ ID NO: 57CDR_(H)2 RIRSKYNNYATYYADSVK X SEQ ID NO: 58 CDR_(H)3 HGNFGNS 

VSWFAY SEQ ID NO: 73 CDR_(L)1 RSSTGAVTTSNYAN SEQ ID NO: 60 CDR_(L)2GTNKRAP SEQ ID NO: 61 CDR_(L)3 ALWYSNLWV SEQ ID NO: 62 wherein X isaspartate (D) or glycine (G)

6. CD3 mAb 1 M6

The amino acid sequence of the VH Domain of CD3 mAb 1 M6 (SEQ ID NO:74)is shown below (CDR_(H) residues are shown underlined). Relative to SEQID NO:55, SEQ ID NO:74 contains a Y100bQ substitution (shown in doubleunderline, and numbered as in Kabat); additionally, position 65,numbered as in Kabat, of SEQ ID NO:74, also shown in double underline,may be aspartate (D) or glycine (G):

EVQLVESGGG LVQPGGSLRL SCAASGFTFS  TYAMN WVRQA PGKGLEWVG R  IRSKYNNYAT YYADSVK  

RF TISRDDSKNS LYLQMNSLKT EDTAVYYCVR  HGNFGNS  

VS WFAY WGQGTL VTVSS

-   -   wherein X is aspartate (D) or glycine (G)

A preferred amino acid sequence of the VL Domain of CD3 mAb 1 M6 is SEQID NO:56.

CD3 mAb 1 M6 CDR Sequence SEQ ID NO CDR_(H)1 TYAMN SEQ ID NO: 57CDR_(H)2 RIRSKYNNYATYYADSVK X SEQ ID NO: 58 CDR_(H)3 HGNFGNS 

VSWFAY SEQ ID NO: 75 CDR_(L)1 RSSTGAVTTSNYAN SEQ ID NO: 60 CDR_(L)2GTNKRAP SEQ ID NO: 61 CDR_(L)3 ALWYSNLWV SEQ ID NO: 62 wherein X isaspartate (D) or glycine (G)

7. CD3 mAb 1 M7

The amino acid sequence of the VH Domain of CD3 mAb 1 M7 (SEQ ID NO:76)is shown below (CDR_(H) residues are shown underlined). Relative to SEQID NO:55, SEQ ID NO:76 contains a G96D substitution (shown in doubleunderline, and numbered as in Kabat); additionally, position 65,numbered as in Kabat, of SEQ ID NO:76, also shown in double underline,may be aspartate (D) or glycine (G):

EVQLVESGGG LVQPGGSLRL SCAASGFTFS  TYAMN WVRQA PGKGLEWVG R  IRSKYNNYAT YYADSVK  

RF TISRDDSKNS LYLQMNSLKT EDTAVYYCVR  H  

NFGNSYVS WFAY WGQGTL VTVSS

-   -   wherein X is aspartate (D) or glycine (G)

A preferred amino acid sequence of the VL Domain of CD3 mAb 1 M7 is SEQID NO:56.

CD3 mAb 1 M7 CDR Sequence SEQ ID NO CDR_(H)1 TYAMN SEQ ID NO: 57CDR_(H)2 RIRSKYNNYATYYADSVK X SEQ ID NO: 58 CDR_(H)3 H 

NFGNSYVSWFAY SEQ ID NO: 77 CDR_(L)1 RSSTGAVTTSNYAN SEQ ID NO: 60CDR_(L)2 GTNKRAP SEQ ID NO: 61 CDR_(L)3 ALWYSNLWV SEQ ID NO: 62 whereinX is aspartate (D) or glycine (G)

8. CD3 mAb 1 M8

The amino acid sequence of the VH Domain of CD3 mAb 1 M8 (SEQ ID NO:78)is shown below (CDR_(H) residues are shown underlined). Relative to SEQID NO:55, SEQ ID NO:78 contains a G99E substitution (shown in doubleunderline, and numbered as in Kabat); additionally, position 65,numbered as in Kabat, of SEQ ID NO:78, also shown in double underline,may be aspartate (D) or glycine (G):

EVQLVESGGG LVQPGGSLRL SCAASGFTFS  TYAMN WVRQA PGKGLEWVGR IRSKYNNYAT YYADSVK  

RF TISRDDSKNS LYLQMNSLKT EDTAVYYCVR  H  

NFGNSYVS WFAY WGQGTL VTVSS

-   -   wherein X is aspartate (D) or glycine (G)

A preferred amino acid sequence of the VL Domain of CD3 mAb 1 M8 is SEQID NO:56.

CD3 mAb 1 M8 CDR Sequence SEQ ID NO CDR_(H)1 TYAMN SEQ ID NO: 57CDR_(H)2 RIRSKYNNYATYYADSVK X SEQ ID NO: 58 CDR_(H)3 H 

NFGNSYVSWFAY SEQ ID NO: 79 CDR_(L)1 RSSTGAVTTSNYAN SEQ ID NO: 60CDR_(L)2 GTNKRAP SEQ ID NO: 61 CDR_(L)3 ALWYSNLWV SEQ ID NO: 62 whereinX is aspartate (D) or glycine (G)

9. CD3 mAb 1 M9

The amino acid sequence of the VH Domain of CD3 mAb 1 M9 (SEQ ID NO:80)is shown below (CDR_(H) residues are shown underlined). Relative to SEQID NO:55, SEQ ID NO:80 contains a G99K substitution (shown in doubleunderline, and numbered as in Kabat); additionally, position 65,numbered as in Kabat, of SEQ ID NO:80, also shown in double underline,may be aspartate (D) or glycine (G)):

EVQLVESGGG LVQPGGSLRL SCAASGFTFS  TYAMN WVRQA PGKGLEWVG R  IRSKYNNYAT YYADSVK  

RF TISRDDSKNS LYLQMNSLKT EDTAVYYCVR  H  

NFGNSYVS WFAY WGQGTL VTVSS

-   -   wherein X is aspartate (D) or glycine (G)

A preferred amino acid sequence of the VL Domain of CD3 mAb 1 M9 is SEQID NO:56.

CD3 mAb 1 M9 CDR Sequence SEQ ID NO CDR_(H)1 TYAMN SEQ ID NO: 57CDR_(H)2 RIRSKYNNYATYYADSVK X SEQ ID NO: 58 CDR_(H)3 H 

NFGNSYVSWFAY SEQ ID NO: 81 CDR_(L)1 RSSTGAVTTSNYAN SEQ ID NO: 60CDR_(L)2 GTNKRAP SEQ ID NO: 61 CDR_(L)3 ALWYSNLWV SEQ ID NO: 62 whereinX is aspartate (D) or glycine (G)

10. CD3 mAb 1 M10

The amino acid sequence of the VH Domain of CD3 mAb 1 M10 (SEQ ID NO:82)is shown below (CDR_(H) residues are shown underlined). Relative to SEQID NO:55, SEQ ID NO:82 contains a F98I substitution (shown in doubleunderline, and numbered as in Kabat); additionally, position 65,numbered as in Kabat, of SEQ ID NO:82, also shown in double underline,may be aspartate (D) or glycine (G):

EVQLVESGGG LVQPGGSLRL SCAASGFTFS  TYAMN WVRQA PGKGLEWVG R  IRSKYNNYAT YYADSVK  

RF TISRDDSKNS LYLQMNSLKT EDTAVYYCVR  HGN  

GNSYVS WFAY WGQGTL VTVSS

-   -   wherein X is aspartate (D) or glycine (G)

A preferred amino acid sequence of the VL Domain of CD3 mAb 1 M10 is SEQID NO:56.

CD3 mAb 1 M10 CDR Sequence SEQ ID NO CDR_(H)1 TYAMN SEQ ID NO: 57CDR_(H)2 RIRSKYNNYATYYADSVK X SEQ ID NO: 58 CDR_(H)3 HGN 

GNSYVSWFAY SEQ ID NO: 83 CDR_(L)1 RSSTGAVTTSNYAN SEQ ID NO: 60 CDR_(L)2GTNKRAP SEQ ID NO: 61 CDR_(L)3 ALWYSNLWV SEQ ID NO: 62 wherein X isaspartate (D) or glycine (G)

11. CD3 mAb 1 M11

The amino acid sequence of the VH Domain of CD3 mAb 1 M11 (SEQ ID NO:84)is shown below (CDR_(H) residues are shown underlined). Relative to SEQID NO:55, SEQ ID NO:84 contains a W100eF substitution (shown in doubleunderline, and numbered as in Kabat); additionally, position 65,numbered as in Kabat, of SEQ ID NO:84, also shown in double underline,may be aspartate (D) or glycine (G):

EVQLVESGGG LVQPGGSLRL SCAASGFTFS  TYAMN WVRQA PGKGLEWVG R  IRSKYNNYAT YYADSVK  

RF TISRDDSKNS LYLQMNSLKT EDTAVYYCVR  HGNFGNSYVS  

FAY WGQGTL VTVSS

-   -   wherein X is aspartate (D) or glycine (G)

A preferred amino acid sequence of the VL Domain of CD3 mAb 1 M11 is SEQID NO:56.

CD3 mAb 1 M11 CDR Sequence SEQ ID NO CDR_(H)1 TYAMN SEQ ID NO: 57CDR_(H)2 RIRSKYNNYATYYADSVK X SEQ ID NO: 58 CDR_(H)3 HGNFGNSYVS 

FAY SEQ ID NO: 85 CDR_(L)1 RSSTGAVTTSNYAN SEQ ID NO: 60 CDR_(L)2 GTNKRAPSEQ ID NO: 61 CDR_(L)3 ALWYSNLWV SEQ ID NO: 62 wherein X is aspartate(D) or glycine (G)

12. CD3 mAb 1 M12

The amino acid sequence of the VH Domain of CD3 mAb 1 M12 (SEQ ID NO:86)is shown below (CDR_(H) residues are shown underlined). Relative to SEQID NO:55, SEQ ID NO:86 contains a W100eY substitution (shown in doubleunderline, and numbered as in Kabat); additionally, position 65,numbered as in Kabat, of SEQ ID NO:86, also shown in double underline,may be aspartate (D) or glycine (G):

EVQLVESGGG LVQPGGSLRL SCAASGFTFS  TYAMN WVRQA PGKGLEWVG R  IRSKYNNYAT YYADSVK  

RF TISRDDSKNS LYLQMNSLKT EDTAVYYCVR  HGNFGNSYVS  

FAY WGQGTL VTVSS

-   -   wherein X is aspartate (D) or glycine (G)

A preferred amino acid sequence of the VL Domain of CD3 mAb 1 M12 is SEQID NO:56.

CD3 mAb 1 M12 CDR Sequence SEQ ID NO CDR_(H)1 TYAMN SEQ ID NO: 57CDR_(H)2 RIRSKYNNYATYYADSVK X SEQ ID NO: 58 CDR_(H)3 HGNFGNSYVS 

FAY SEQ ID NO: 87 CDR_(L)1 RSSTGAVTTSNYAN SEQ ID NO: 60 CDR_(L)2 GTNKRAPSEQ ID NO: 61 CDR_(L)3 ALWYSNLWV SEQ ID NO: 62 wherein X is aspartate(D) or glycine (G)

13. CD3 mAb 1 M13

The amino acid sequence of the VH Domain of CD3 mAb 1 M13 (SEQ ID NO:88)is shown below (CDR_(H) residues are shown underlined). Relative to SEQID NO:55, SEQ ID NO:88 contains a Y102E substitution (shown in doubleunderline, and numbered as in Kabat); additionally, position 65,numbered as in Kabat, of SEQ ID NO:88, also shown in double underline,may be aspartate (D) or glycine (G):

EVQLVESGGG LVQPGGSLRL SCAASGFTFS  TYAMN WVRQA PGKGLEWVG R  IRSKYNNYAT YYADSVK  

RF TISRDDSKNS LYLQMNSLKT EDTAVYYCVR  HGNFGNSYVS WFA  

WGQGTL VTVSS

-   -   wherein X is aspartate (D) or glycine (G)

A preferred amino acid sequence of the VL Domain of CD3 mAb 1 M13 is SEQID NO:56.

CD3 mAb 1 M13 CDR Sequence SEQ ID NO CDR_(H)1 TYAMN SEQ ID NO: 57CDR_(H)2 RIRSKYNNYATYYADSVK X SEQ ID NO: 58 CDR_(H)3 HGNFGNSYVSWFA 

SEQ ID NO: 89 CDR_(L)1 RSSTGAVTTSNYAN SEQ ID NO: 60 CDR_(L)2 GTNKRAPSEQ ID NO: 61 CDR_(L)3 ALWYSNLWV SEQ ID NO: 62 wherein X is aspartate(D) or glycine (G)

14. CD3 mAb 1 M14

The amino acid sequence of the VH Domain of CD3 mAb 1 M14 (SEQ ID NO:90)is shown below (CDR_(H) residues are shown underlined). Relative to SEQID NO:55, SEQ ID NO:90 contains a T31D substitution (shown in doubleunderline, and numbered as in Kabat); additionally, position 65,numbered as in Kabat, of SEQ ID NO:90, also shown in double underline,may be aspartate (D) or glycine (G):

EVQLVESGGG LVQPGGSLRL SCAASGFTFS 

YAMN WVRQA PGKGLEWVG R   IRSKYNNYAT YYADSVK  

RF TISRDDSKNS LYLQMNSLKT EDTAVYYCVR  HGNFGNSYVS WFAY WGQGTL VTVSS

-   -   wherein X is aspartate (D) or glycine (G)

A preferred amino acid sequence of the VL Domain of CD3 mAb 1 M14 is SEQID NO:56.

CD3 mAb 1 M14 CDR Sequence SEQ ID NO CDR_(H)1

YAMN SEQ ID NO: 91 CDR_(H)2 RIRSKYNNYATYYADSVK X SEQ ID NO: 58 CDR_(H)3HGNFGNSYVSWFAY SEQ ID NO: 59 CDR_(L)1 RSSTGAVTTSNYAN SEQ ID NO: 60CDR_(L)2 GTNKRAP SEQ ID NO: 61 CDR_(L)3 ALWYSNLWV SEQ ID NO: 62 whereinX is aspartate (D) or glycine (G)

15. CD3 mAb 1 M15

The amino acid sequence of the VH Domain of CD3 mAb 1 M15 (SEQ ID NO:92)is shown below (CDR_(H) residues are shown underlined). Relative to SEQID NO:55, SEQ ID NO:92 contains a T33E substitution (shown in doubleunderline, and numbered as in Kabat); additionally, position 65,numbered as in Kabat, of SEQ ID NO:92, also shown in double underline,may be aspartate (D) or glycine (G):

EVQLVESGGG LVQPGGSLRL SCAASGFTFS 

YAMN WVRQA PGKGLEWVG R   IRSKYNNYAT YYADSVK  

RF TISRDDSKNS LYLQMNSLKT EDTAVYYCVR  HGNFGNSYVS WFAY WGQGTL VTVSS

-   -   wherein X is aspartate (D) or glycine (G)

A preferred amino acid sequence of the VL Domain of CD3 mAb 1 M15 is SEQID NO:56.

CD3 mAb 1 M15 CDR Sequence SEQ ID NO CDR_(H)1

YAMN SEQ ID NO: 93 CDR_(H)2 RIRSKYNNYATYYADSVK X SEQ ID NO: 58 CDR_(H)3HGNFGNSYVSWFAY SEQ ID NO: 59 CDR_(L)1 RSSTGAVTTSNYAN SEQ ID NO: 60CDR_(L)2 GTNKRAP SEQ ID NO: 61 CDR_(L)3 ALWYSNLWV SEQ ID NO: 62 whereinX is aspartate (D) or glycine (G)

16. CD3 mAb 1 M16

The amino acid sequence of the VH Domain of CD3 mAb 1 M16 (SEQ ID NO:94)is shown below (CDR_(H) residues are shown underlined). Relative to SEQID NO:55, SEQ ID NO:94 contains a Y32D substitution (shown in doubleunderline, and numbered as in Kabat); additionally, position 65,numbered as in Kabat, of SEQ ID NO:94, also shown in double underline,may be aspartate (D) or glycine (G):

EVQLVESGGG LVQPGGSLRL SCAASGFTFS  T  

AMN WVRQA PGKGLEWVG R   IRSKYNNYAT YYADSVK  

RF TISRDDSKNS LYLQMNSLKT EDTAVYYCVR  HGNFGNSYVS WFAY WGQGTL VTVSS

-   -   wherein X is aspartate (D) or glycine (G)

A preferred amino acid sequence of the VL Domain of CD3 mAb 1 M16 is SEQID NO:56.

CD3 mAb 1 M16 CDR Sequence SEQ ID NO CDR_(H)1 T 

AMN SEQ ID NO: 95 CDR_(H)2 RIRSKYNNYATYYADSVK X SEQ ID NO: 58 CDR_(H)3HGNFGNSYVSWFAY SEQ ID NO: 59 CDR_(L)1 RSSTGAVTTSNYAN SEQ ID NO: 60CDR_(L)2 GTNKRAP SEQ ID NO: 61 CDR_(L)3 ALWYSNLWV SEQ ID NO: 62 whereinX is aspartate (D) or glycine (G)

17. CD3 mAb 1 M17

The amino acid sequence of the VH Domain of CD3 mAb 1 M17 (SEQ ID NO:96)is shown below (CDR_(H) residues are shown underlined). Relative to SEQID NO:55, SEQ ID NO:96 contains a Y32T substitution (shown in doubleunderline, and numbered as in Kabat); additionally, position 65,numbered as in Kabat, of SEQ ID NO:96, also shown in double underline,may be aspartate (D) or glycine (G):

EVQLVESGGG LVQPGGSLRL SCAASGFTFS  T  

AMN WVRQA PGKGLEWVG R   IRSKYNNYAT YYADSVK  

RF TISRDDSKNS LYLQMNSLKT EDTAVYYCVR  HGNFGNSYVS WFAY WGQGTL VTVSS

-   -   wherein X is aspartate (D) or glycine (G)

A preferred amino acid sequence of the VL Domain of CD3 mAb 1 M17 is SEQID NO:56.

CD3 mAb 1 M17 CDR Sequence SEQ ID NO CDR_(H)1 T 

TAMN SEQ ID NO: 97 CDR_(H)2 RIRSKYNNYATYYADSVK X SEQ ID NO: 58 CDR_(H)3HGNFGNSYVSWFAY SEQ ID NO: 59 CDR_(L)1 RSSTGAVTTSNYAN SEQ ID NO: 60CDR_(L)2 GTNKRAP SEQ ID NO: 61 CDR_(L)3 ALWYSNLWV SEQ ID NO: 62 whereinX is aspartate (D) or glycine (G)

18. CD3 mAb 1 M18

The amino acid sequence of the VH Domain of CD3 mAb 1 M18 (SEQ ID NO:98)is shown below (CDR_(H) residues are shown underlined). Relative to SEQID NO:55, SEQ ID NO:98 contains a A33G substitution (shown in doubleunderline, and numbered as in Kabat); additionally, position 65,numbered as in Kabat, of SEQ ID NO:98, also shown in double underline,may be aspartate (D) or glycine (G):

EVQLVESGGG LVQPGGSLRL SCAASGFTFS  TY  

MN WVRQA PGKGLEWVGR  IRSKYNNYAT   YYADSVK  

RF TISRDDSKNS LYLQMNSLKT EDTAVYYCVR  HGNFGNSYVS   WFAY WGQGTL VTVSS

-   -   wherein X is aspartate (D) or glycine (G)

A preferred amino acid sequence of the VL Domain of CD3 mAb 1 M18 is SEQID NO:56.

CD3 mAb 1 M18 CDR Sequence SEQ ID NO CDR_(H)1 TY 

MN SEQ ID NO: 99 CDR_(H)2 RIRSKYNNYATYYADSVK X SEQ ID NO: 58 CDR_(H)3HGNFGNSYVSWFAY SEQ ID NO: 59 CDR_(L)1 RSSTGAVTTSNYAN SEQ ID NO: 60CDR_(L)2 GTNKRAP SEQ ID NO: 61 CDR_(L)3 ALWYSNLWV SEQ ID NO: 62 whereinX is aspartate (D) or glycine (G)

19. CD3 mAb 1 M19

The amino acid sequence of the VH Domain of CD3 mAb 1 M19 (SEQ IDNO:100) is shown below (CDR_(H) residues are shown underlined). Relativeto SEQ ID NO:55, SEQ ID NO:100 contains G96K and F98I substitutions(shown in double underline, and numbered as in Kabat); additionally,position 65, numbered as in Kabat, of SEQ ID NO:100, also shown indouble underline, may be aspartate (D) or glycine (G):

EVQLVESGGG LVQPGGSLRL SCAASGFTFS  TYAMN WVRQA PGKGLEWVG R  IRSKYNNYAT YYADSVK  

RF TISRDDSKNS LYLQMNSLKT EDTAVYYCVR  H  

N  

GNSYVS WFAY WGQGTL VTVSS

-   -   wherein X is aspartate (D) or glycine (G)

A preferred amino acid sequence of the VL Domain of CD3 mAb 1 M19 is SEQID NO:56.

CD3 mAb 1 M19 CDR Sequence SEQ ID NO CDR_(H)1 TYAMN SEQ ID NO: 57CDR_(H)2 RIRSKYNNYATYYADSVK X SEQ ID NO: 58 CDR_(H)3 H 

N 

GNSYVSWFAY SEQ ID NO: 101 CDR_(L)1 RSSTGAVTTSNYAN SEQ ID NO: 60 CDR_(L)2GTNKRAP SEQ ID NO: 61 CDR_(L)3 ALWYSNLWV SEQ ID NO: 62 wherein X isaspartate (D) or glycine (G)

20. CD3 mAb 1 M20

The amino acid sequence of the VH Domain of CD3 mAb 1 M20 (SEQ IDNO:102) is shown below (CDR_(H) residues are shown underlined). Relativeto SEQ ID NO:55, SEQ ID NO:102 contains G96K and Y100bG substitutions(shown in double underline, and numbered as in Kabat); additionally,position 65, numbered as in Kabat, of SEQ ID NO:102, also shown indouble underline, may be aspartate (D) or glycine (G):

EVQLVESGGG LVQPGGSLRL SCAASGFTFS  TYAMN WVRQA PGKGLEWVG R  IRSKYNNYAT YYADSVK  

RF TISRDDSKNS LYLQMNSLKT EDTAVYYCVR  H  

NFGNS 

VS WFAY WGQGTL VTVSS

-   -   wherein X is aspartate (D) or glycine (G)

A preferred amino acid sequence of the VL Domain of CD3 mAb 1 M20 is SEQID NO:56.

CD3 mAb 1 M20 CDR Sequence SEQ ID NO CDR_(H)1 TYAMN SEQ ID NO: 57CDR_(H)2 RIRSKYNNYATYYADSVK X SEQ ID NO: 58 CDR_(H)3 H 

NFGNS 

VSWFAY SEQ ID NO: 103 CDR_(L)1 RSSTGAVTTSNYAN SEQ ID NO: 60 CDR_(L)2GTNKRAP SEQ ID NO: 61 CDR_(L)3 ALWYSNLWV SEQ ID NO: 62 wherein X isaspartate (D) or glycine (G)

21. CD3 mAb 1 M21

The amino acid sequence of the VH Domain of CD3 mAb 1 M21 (SEQ IDNO:104) is shown below (CDR_(H) residues are shown underlined). Relativeto SEQ ID NO:55, SEQ ID NO:104 contains G96K and W 100eF substitutions(shown in double underline, and numbered as in Kabat); additionally,position 65, numbered as in Kabat, of SEQ ID NO: 104, also shown indouble underline, may be aspartate (D) or glycine (G):

EVQLVESGGG LVQPGGSLRL SCAASGFTFS  TYAM NWVRQA PGKGLEWVG R  IRSKYNNYAT YYADSVK  

RF TISRDDSKNS LYLQMNSLKT EDTAVYYCVR  H  

NFGNSYVS 

FAY WGQGTL VTVSS

-   -   wherein X is aspartate (D) or glycine (G)

A preferred amino acid sequence of the VL Domain of CD3 mAb 1 M21 is SEQID NO:56.

CD3 mAb 1 M21 CDR Sequence SEQ ID NO CDR_(H)1 TYAMN SEQ ID NO: 57CDR_(H)2 RIRSKYNNYATYYADSVK X SEQ ID NO: 58 CDR_(H)3 H 

NFGNSYVS 

FAY SEQ ID NO: 105 CDR_(L)1 RSSTGAVTTSNYAN SEQ ID NO: 60 CDR_(L)2GTNKRAP SEQ ID NO: 61 CDR_(L)3 ALWYSNLWV SEQ ID NO: 62 wherein X isaspartate (D) or glycine (G)

22. CD3 mAb 1 M22

The amino acid sequence of the VH Domain of CD3 mAb 1 M22 (SEQ IDNO:106) is shown below (CDR_(H) residues are shown underlined). Relativeto SEQ ID NO:55, SEQ ID NO:106 contains G96K and W100eY substitutions(shown in double underline, and numbered as in Kabat); additionally,position 65, numbered as in Kabat, of SEQ ID NO:106, also shown indouble underline, may be aspartate (D) or glycine (G):

EVQLVESGGG LVQPGGSLRL SCAASGFTFS  TYAMN WVRQA PGKGLEWVG R  IRSKYNNYAT YYADSVK  

RF TISRDDSKNS LYLQMNSLKT EDTAVYYCVR  H  

NFGNSYVS 

FAY WGQGTL VTVSS

-   -   wherein X is aspartate (D) or glycine (G)

A preferred amino acid sequence of the VL Domain of CD3 mAb 1 M22 is SEQID NO:56.

CD3 mAb 1 M22 CDR Sequence SEQ ID NO CDR_(H)1 TYAMN SEQ ID NO: 57CDR_(H)2 RIRSKYNNYATYYADSVK X SEQ ID NO: 58 CDR_(H)3 H 

NFGNSYVS 

FAY SEQ ID NO: 107 CDR_(L)1 RSSTGAVTTSNYAN SEQ ID NO: 60 CDR_(L)2GTNKRAP SEQ ID NO: 61 CDR_(L)3 ALWYSNLWV SEQ ID NO: 62 wherein X isaspartate (D) or glycine (G)

23. CD3 mAb 1 M23

A preferred amino acid sequence of the VH Domain of CD3 mAb 1 M23 is SEQID NO:55 or SEQ ID NO:63.

The amino acid sequence of the VL Domain of CD3 mAb 1 M23 (SEQ IDNO:108) is shown below (CDR_(L) residues are shown underlined). Relativeto SEQ ID NO:56, SEQ ID NO:108 contains an L95E substitution (shown indouble underline, and numbered as in Kabat):

QAVVTQEPSL TVSPGGTVTL TC RSSTGAVT TSNYAN WVQQ KPGQAPRGLI G GTNKRAPWT PARFSGSLLG GKAALTITGA QAEDEADYYC  A  

WYSNLWV F GGGTKLTVLG

CD3 mAb 1 M23 CDR Sequence SEQ ID NO CDR_(H)1 TYAMN SEQ ID NO: 57CDR_(H)2 RIRSKYNNYATYYADSVK X SEQ ID NO: 58 CDR_(H)3 HGNFGNSYVSWFAYSEQ ID NO: 59 CDR_(L)1 RSSTGAVTTSNYAN SEQ ID NO: 60 CDR_(L)2 GTNKRAPSEQ ID NO: 61 CDR_(L)3 A 

WYSNLWV SEQ ID NO: 109 wherein X is aspartate (D) or glycine (G)

24. CD3 mAb 1 M24

A preferred amino acid sequence of the VH Domain of CD3 mAb 1 M24 is SEQID NO:55 or SEQ ID NO:63.

The amino acid sequence of the VL Domain of CD3 mAb 1 M24 (SEQ IDNO:11R) is shown below (CDR_(L) residues are shown underlined). Relativeto SEQ ID NO:56, SEQ ID NO:115 contains an L95Q substitution (shown indouble underline, and numbered as in Kabat):

QAVVIQEPSL TVSPGGTVTL TC RSSTGAVT TSNYAN WVQQ KPGQAPRGLI G GTNKRAPWT PARFSGSLLG GKAALTITGA QAEDEADYYC  A 

WYSNLWV F GGGTKLIVLG

CD3 mAb 1 M24 CDR Sequence SEQ ID NO CDR_(H)1 TYAMN SEQ ID NO: 57CDR_(H)2 RIRSKYNNYATYYADSVK X SEQ ID NO: 58 CDR_(H)3 HGNFGNSYVSWFAYSEQ ID NO: 59 CDR_(L)1 RSSTGAVTTSNYAN SEQ ID NO: 60 CDR_(L)2 GTNKRAPSEQ ID NO: 61 CDR_(L)3 A 

WYSNLWV SEQ ID NO: 111 wherein X is aspartate (D) or glycine (G)

25. CD3 mAb 1 M25

A preferred amino acid sequence of the VH Domain of CD3 mAb 1 M25 is SEQID NO:55 or SEQ ID NO:63.

The amino acid sequence of the VL Domain of CD3 mAb 1 M25 (SEQ IDNO:112) is shown below (CDR_(L) residues are shown underlined). Relativeto SEQ ID NO:56, SEQ ID NO:112 contains a G50D substitution (shown indouble underline, and numbered as in Kabat):

QAVVTQEPSL TVSPGGTVTL TC RSSTGAVT TSNYAN WVQQ KPGQAPRGLI G 

TNKRAP WT PARFSGSLLG GKAALTITGA QAEDEADYYC  ALWYSNLWV F GGGTKLIVLG

CD3 mAb 1 M25 CDR Sequence SEQ ID NO CDR_(H)1 TYAMN SEQ ID NO: 57CDR_(H)2 RIRSKYNNYATYYADSVK X SEQ ID NO: 58 CDR_(H)3 HGNFGNSYVSWFAYSEQ ID NO: 59 CDR_(L)1 RSSTGAVTTSNYAN SEQ ID NO: 60 CDR_(L)2

TNKRAP SEQ ID NO: 113 CDR_(L)3 ALWYSNLWV SEQ ID NO: 62 wherein X isaspartate (D) or glycine (G)

26. CD3 mAb 1 M26

A preferred amino acid sequence of the VH Domain of CD3 mAb 1 M26 is SEQID NO:55 or SEQ ID NO:63.

The amino acid sequence of the VL Domain of CD3 mAb 1 M26 (SEQ IDNO:114) is shown below (CDR_(L) residues are shown underlined). Relativeto SEQ ID NO:56, SEQ ID NO:114 contains a K53G substitution (shown indouble underline):

QAVVIQEPSL TVSPGGTVTL TC RSSTGAVT TSNYAN WVQQ KPGQAPRGLI G GTN  

RAP WT PARFSGSLLG GKAALTITGA QAEDEADYYC  ALWYSNLWV F GGGTKLTVLG

CD3 mAb 1 M26 CDR Sequence SEQ ID NO CDR_(H)1 TYAMN SEQ ID NO: 57CDR_(H)2 RIRSKYNNYATYYADSVK X SEQ ID NO: 58 CDR_(H)3 HGNFGNSYVSWFAYSEQ ID NO: 59 CDR_(L)1 RSSTGAVTTSNYAN SEQ ID NO: 60 CDR_(L)2 GTN 

RAP SEQ ID NO: 115 CDR_(L)3 ALWYSNLWV SEQ ID NO: 62 wherein X isaspartate (D) or glycine (G)

C. Exemplary Antibodies that Bind to Cell Surface Molecules of anEffector Cell

As used herein, the term “effector cell” denotes a cell that directly orindirectly mediates the killing of target cells (e.g., foreign cells,infected cells or cancer cells). Examples of effector cells includehelper T-cells, cytotoxic T-cells, Natural Killer (NK) cells, plasmacells (antibody-secreting B cells), macrophages and granulocytes.Preferred cell surface molecules of such cells include CD2, CD3, CD8,CD16, TCR, and the NKG2D receptor. Accordingly, molecules capable ofimmunospecifically binding an epitope of such molecules, or to othereffector cell surface molecules may be used in accordance with theprinciples of the present invention. Exemplary antibodies, whose VH andVL Domains may be used to construct molecules capable of mediating theredirected killing of a target cell are provided below.

1. Exemplary Anti-CD2 Antibodies

In one embodiment, the molecules of the present invention that arecapable of mediating the redirected killing of a target cell will bindan effector cell by immunospecifically binding an epitope of CD2 presenton the surface of such effector cell. Molecules that specifically bindCD2 include the anti-CD2 antibody “CD2 mAb Lo-CD2a.”

The amino acid sequence of the VH Domain of CD2 mAb Lo-CD2a (ATCCAccession No: 11423); SEQ ID NO:116) is shown below (CDR_(H) residuesare shown underlined):

EVQLQQSGPE LQRPGASVKL SCKASGYIFT  EYYMY WVKQR PKQGLELVG R   IDPEDGSIDY  VEKFKK KATL TADTSSNTAY MQLSSLTSED TATYFCAR GK   FNYRFAY WGQ GTLVTVSS

The amino acid sequence of the VL Domain of CD2 mAb Lo-CD2a (ATCCAccession No: 11423; SEQ ID NO:117) is shown below (CDR_(L) residues areshown underlined):

DVVLTQTPPT LLATIGQSVS ISC RSSQSLL   HSSGNTYLN W LLQRTGQSPQ PLIY LVSKLE  S GVPNRFSGS GSGTDFTLKI SGVEAEDLGV YYC MQFTHYP   YT FGAGTKLE LK

2. Exemplary Anti-CD8 Antibodies

In one embodiment, the molecules of the present invention that arecapable of mediating the redirected killing of a target cell will bindan effector cell by immunospecifically binding an epitope of CD8 presenton the surface of such effector cell. Antibodies that specifically bindCD8 include the anti-CD8 antibodies “OKT8” and “TRX2.”

The amino acid sequence of the VH Domain of OKT8 (SEQ ID NO:118) isshown below (CDR_(H) residues are shown underlined):

QVQLLESGPE LLKPGASVKM SCKA SGYTFT   DYNMH WVKQS HGKSLEWIG Y   IYPYTGGTGY  NQKFKN KATL TVDSSSSTAY MELRSLTSED SAVYYCARNF RYTYWYFDVW GQGTTVTVSS

The amino acid sequence of the VL Domain of OKT8 (SEQ ID NO:119) isshown below (CDR_(L) residues are shown underlined):

DIVMTQSPAS LAVSLGQRAT ISCRASESVD  SYDNSLMH WY QQKPGQPPKV LIY LASNLES GVPARFSGSG SRTDFTLTID PVEADDAATY YC QQNNEDPY   T FGGGTKLEI KR

The amino acid sequence of the VH Domain of TRX2 (SEQ ID NO:120) isshown below (CDR_(H) residues are shown underlined):

QVQLVESGGG VVQPGRSLRL SCAASGFTFS  DFGMN WVRQA PGKGLEWVA L   IYYDGSNKFY  ADSVKG RFTI SRDNSKNTLY LQMNSLRAED TAVYYCAK PH   YDGYYHFFDS  WGQGTLVTVS S

The amino acid sequence of the VL Domain of TRX2 (SEQ ID NO:121) isshown below (CDR_(L) residues are shown underlined):

DIQMTQSPSS LSASVGDRVT ITC KGSQDIN   NYLA WYQQKP GKAPKLLIY N   TDILHTGVPS RFSGSGSGTD FTFTISSLQP EDIATYYC YQ YNNGYT FGQG TKVEIK

VI. Exemplary Cancer and Pathogen-Associated Antigens

A. Exemplary Cancer Antigens Arrayed on the Surface of Cancer Cells

As used herein, the term “Cancer Antigen” denotes an antigen that ischaracteristically expressed on the surface of a cancer cell, and thatmay thus be treated with an Antibody-Based Molecule or anImmunomodulatory Molecule. Examples of Cancer Antigens include, but arenot limited to: 19.9 as found in colon cancer, gastric cancer mucins;4.2; ADAM-9 (United States Patent Publication No. 2006/0172350; PCTPublication No. WO 06/084075); AH6 as found in gastric cancer; ALCAM(PCT Publication No. WO 03/093443); APO-1 (malignant human lymphocyteantigen) (Trauth, B. C. et al. (1989) “Monoclonal Antibody-MediatedTumor Regression By Induction Of Apoptosis,” Science 245:301-304); B1(Egloff, A. M. et al. (2006) “Cyclin B1 And Other Cyclins As TumorAntigens In Immunosurveillance And Immunotherapy Of Cancer,” Cancer Res.66(1):6-9); B7-H3 (Collins, M. et al. (2005) “The B7 Family OfImmune-Regulatory Ligands,” Genome Biol. 6:223.1-223.7). Chapoval, A. etal. (2001) “B7-H3: A Costimulatory Molecule For T Cell Activation andIFN-γ Production,” Nature Immunol. 2:269-274; Sun, M. et al. (2002)“Characterization of Mouse and Human B7-H3 Genes,” J. Immunol.168:6294-6297); BAGE (Bodey, B. (2002) “Cancer-Testis Antigens:Promising Targets For Antigen Directed Antineoplastic Immunotherapy,”Expert Opin. Biol. Ther. 2(6):577-584); beta-catenin (Prange W. et al.(2003) “Beta-Catenin Accumulation In The Progression Of HumanHepatocarcinogenesis Correlates With Loss Of E-Cadherin And AccumulationOf P53, But Not With Expression Of Conventional WNT-1 Target Genes,” J.Pathol. 201(2):250-259); blood group ALe^(b)/Le^(y) as found in colonicadenocarcinoma; Burkitt's lymphoma antigen-38.13; C14 as found incolonic adenocarcinoma; CA125 (ovarian carcinoma antigen) (Bast, R. C.Jr. et al. (2005) “New Tumor Markers: CA125 And Beyond,” Int. J.Gynecol. Cancer 15(Suppl 3):274-281; Yu et al. (1991) “Coexpression OfDifferent Antigenic Markers On Moieties That Bear CA 125 Determinants,”Cancer Res. 51(2):468-475); Carboxypeptidase M (United States PatentPublication No. 2006/0166291); CD5 (Calin, G. A. et al. (2006) “GenomicsOf Chronic Lymphocytic Leukemia MicroRNAs As New Players With ClinicalSignificance,” Semin. Oncol. 33(2):167-173; CD19 (Ghetie et al. (1994)“Anti-CD19 Inhibits The Growth Of Human B-Cell Tumor Lines In Vitro AndOf Daudi Cells In SCID Mice By Inducing Cell Cycle Arrest,” Blood83:1329-1336; Troussard, X. et al. 1998 Hematol Cell Ther.40(4):139-48); CD20 (Reff et al. (1994) “Depletion Of B Cells In Vivo ByA Chimeric Mouse Human Monoclonal Antibody To CD20,” Blood 83:435-445;Thomas, D. A. et al. 2006 Hematol Oncol Clin North Am. 20(5):1125-36);CD22 (Kreitman, R. J. (2006) “Immunotoxins For Targeted Cancer Therapy,”AAPS J. 8(3):E532-51); CD23 (Rosati, S. et al. (2005) “ChronicLymphocytic Leukaemia: A Review Of The Immuno-Architecture,” Curr. Top.Microbiol. Immunol. 294:91-107); CD25 (Troussard, X. et al. (1998)“Hairy Cell Leukemia. What Is New Forty Years After The FirstDescription?” Hematol. Cell. Ther. 40(4):139-148); CD27 (Bataille, R.(2006) “The Phenotype Of Normal, Reactive And Malignant Plasma Cells.Identification Of “Many And Multiple Myelomas” And Of New Targets ForMyeloma Therapy,” Haematologica 91(9):1234-1240); CD28 (Bataille, R.(2006) “The Phenotype Of Normal, Reactive And Malignant Plasma Cells.Identification Of “Many And Multiple Myelomas” And Of New Targets ForMyeloma Therapy,” Haematologica 91(9):1234-1240); CD33 (Sgouros et al.(1993) “Modeling And Dosimetry Of Monoclonal Antibody M195 (Anti-CD33)In Acute Myelogenous Leukemia,” J. Nucl. Med. 34:422-430); CD36 (Ge, Y.(2005) “CD36: A Multiligand Molecule,” Lab Hematol. 11(1):31-7);CD40/CD154 (Messmer, D. et al. (2005) “CD154 Gene Therapy For HumanB-Cell Malignancies,” Ann. N. Y. Acad. Sci. 1062:51-60); CD45 (Jurcic,J. G. (2005) “Immunotherapy For Acute Myeloid Leukemia,” Curr. Oncol.Rep. 7(5):339-346); CD56 (Bataille, R. (2006) “The Phenotype Of Normal,Reactive And Malignant Plasma Cells. Identification Of “Many AndMultiple Myelomas” And Of New Targets For Myeloma Therapy,”Haematologica 91(9):1234-1240); CD46 (U.S. Pat. No. 7,148,038; PCTPublication No. WO 03/032814); CD52 (Eketorp, S. S. et al. (2014)“Alemtuzumab (Anti-CD52 Monoclonal Antibody) As Single-Agent Therapy InPatients With Relapsed/Refractory Chronic Lymphocytic Leukaemia (CLL)-ASingle Region Experience On Consecutive Patients,” Ann Hematol.93(10):1725-1733; Suresh, T. et al. (2014) “New Antibody Approaches ToLymphoma Therapy,” J. Hematol. Oncol. 7:58; Hoelzer, D. (2013) “TargetedTherapy With Monoclonal Antibodies In Acute Lymphoblastic Leukemia,”Curr. Opin. Oncol. 25(6):701-706); CD56 (Bataille, R. (2006) “ThePhenotype Of Normal, Reactive And Malignant Plasma Cells. IdentificationOf “Many And Multiple Myelomas” And Of New Targets For Myeloma Therapy,”Haematologica 91(9):1234-1240); CD79a/CD79b (Troussard, X. et al. (1998)“Hairy Cell Leukemia. What Is New Forty Years After The FirstDescription?” Hematol. Cell. Ther. 40(4):139-148; Chu, P. G. et al.(2001) “CD79: A Review,” Appl. Immunohistochem. Mol. Morphol.9(2):97-106); CD103 (Troussard, X. et al. (1998) “Hairy Cell Leukemia.What Is New Forty Years After The First Description?” Hematol. Cell.Ther. 40(4):139-148); CD317 (Kawai, S. et al. (2008) “Interferon-AEnhances CD317 Expression And The Antitumor Activity Of Anti-CD317Monoclonal Antibody In Renal Cell Carcinoma Xenograft Models,” CancerScience 99(12):2461-2466; Wang, W. et al. (2009) HM1.24 (CD317) Is ANovel Target Against Lung Cancer For Immunotherapy Using Anti-HM1.24Antibody,” Cancer Immunology, Immunotherapy 58(6):967-976; Wang, W. etal. (2009) “Chimeric And Humanized Anti-HM1.24 Antibodies MediateAntibody-Dependent Cellular Cytotoxicity Against Lung Cancer Cells. LungCancer,” 63(1):23-31; Sayeed, A. et al. (2013) “Aberrant Regulation OfThe BST2 (Tetherin) Promoter Enhances Cell Proliferation And ApoptosisEvasion In High Grade Breast Cancer Cells,” PLoS ONE 8(6)e67191, pp.1-10); CDK4 (Lee, Y. M. et al. (2006) “Targeting Cyclins AndCyclin-Dependent Kinases In Cancer: Lessons From Mice, Hopes ForTherapeutic Applications In Human,” Cell Cycle 5(18):2110-2114); CEA(carcinoembryonic antigen; Foon et al. (1995) “Immune Response To TheCarcinoembryonic Antigen In Patients Treated With An Anti-IdiotypeAntibody Vaccine,” J. Clin. Invest. 96(1):334-42); Mathelin, C. (2006)“Circulating Proteinic Biomarkers And Breast Cancer,” Gynecol. Obstet.Fertil. 34(7-8):638-646; Tellez-Avila, F. I. et al. (2005) “TheCarcinoembryonic Antigen: Apropos Of An Old Friend,” Rev. Invest. Clin.57(6):814-819); CEACAM5/CEACAM6 (Zheng, C. et al. (2011) “A NovelAnti-CEACAM5 Monoclonal Antibody, CC4, Suppresses Colorectal TumorGrowth and Enhances NK Cells-Mediated Tumor Immunity,” PLoS One6(6):e21146, pp. 1-11); C017-1A (Ragnhammar et al. (1993) “Effect OfMonoclonal Antibody 17-1A And GM-CSF In Patients With AdvancedColorectal Carcinoma—Long-Lasting, Complete Remissions Can Be Induced,”Int. J. Cancer 53:751-758); CO-43 (blood group Le^(b)); CO-514 (bloodgroup Le^(a)) as found in adenocarcinoma; CTA-1; CTLA-4 (Peggs, K. S. etal. (2006) “Principles And Use Of Anti-CTLA4 Antibody In Human CancerImmunotherapy,” Curr. Opin. Immunol. 18(2):206-13); Cytokeratin 8 (PCTPublication No. WO 03/024191); D1.1; D156-22; DR5 (Abdulghani, J. et al.(2010) “TRAIL Receptor Signaling And Therapeutics,” Expert Opin. Ther.Targets 14(10):1091-1108; Andera, L. (2009) “Signaling Activated By TheDeath Receptors Of The TNFR Family,” Biomed. Pap. Med. Fac. Univ.Palacky Olomouc Czech. Repub. 153(3):173-180; Carlo-Stella, C. et al.(2007) “Targeting TRAIL Agonistic Receptors for Cancer Therapy,” Clin,Cancer 13(8):2313-2317; Chaudhari, B. R. et al. (2006) “Following theTRAIL to Apoptosis,” Immunologic Res. 35(3):249-262); E₁ series (bloodgroup B) as found in pancreatic cancer; EGFR (Epidermal Growth FactorReceptor; Adenis, A. et al. (2003) “Inhibitors Of Epidermal GrowthFactor Receptor And Colorectal Cancer,” Bull. Cancer. 90 Spec No:S228-S232); Ephrin receptors (and in particular EphA2 (U.S. Pat. No.7,569,672; PCT Publication No. WO 06/084226); Erb (ErbB1; ErbB3; ErbB4;Zhou, H. et al. (2002) “Lung Tumorigenesis Associated With Erb-B-2 AndErb-B-3 Overexpression In Human Erb-B-3 Transgenic Mice Is Enhanced ByMethylnitrosourea,” Oncogene 21(57):8732-8740; Rimon, E. et al. (2004)“Gonadotropin-Induced Gene Regulation In Human Granulosa Cells ObtainedFrom IVF Patients: Modulation Of Genes Coding For Growth Factors AndTheir Receptors And Genes Involved In Cancer And Other Diseases,” Int.J. Oncol. 24(5):1325-1338); GAGE (GAGE-1; GAGE-2; Akcakanat, A. et al.(2006) “Heterogeneous Expression Of GAGE, NY-ESO-1, MAGE-A and SSXProteins In Esophageal Cancer: Implications For Immunotherapy,” Int. J.Cancer. 118(1):123-128); GD2/GD3/GM2 (Livingston, P. O. et al. (2005)“Selection Of GM2, Fucosyl GM1, Globo H And Polysialic Acid As TargetsOn Small Cell Lung Cancers For Antibody-Mediated Immunotherapy,” CancerImmunol. Immunother. 54(10):1018-1025); ganglioside GD2 (GD2; Saleh etal. (1993) “Generation Of A Human Anti-Idiotypic Antibody That MimicsThe GD2 Antigen,” J. Immunol., 151, 3390-3398); ganglioside GD3 (G_(D3);Shitara et al. (1993) “A Mouse Human Chimeric Anti-(Ganglioside GD3)Antibody With Enhanced Antitumor Activities,” Cancer Immunol.Immunother. 36:373-380); ganglioside GM2 (G_(M2); Livingston et al.(1994) “Improved Survival In Stage III Melanoma Patients With GM2Antibodies: A Randomized Trial Of Adjuvant Vaccination With GM2Ganglioside,” J. Clin. Oncol. 12:1036-1044); ganglioside GM3 (G_(M3);Hoon et al. (1993) “Molecular Cloning Of A Human Monoclonal AntibodyReactive To Ganglioside GM3 Antigen On Human Cancers,” Cancer Res.53:5244-5250); GICA 19-9 (Herlyn et al. (1982) “Monoclonal AntibodyDetection Of A Circulating Tumor-Associated Antigen. L Presence OfAntigen In Sera Of Patients With Colorectal, Gastric, And PancreaticCarcinoma,” J. Clin. Immunol. 2:135-140); gp100 (Lotem, M. et al. (2006)“Presentation Of Tumor Antigens By Dendritic Cells Genetically ModifiedWith Viral And Nonviral Vectors,” J. Immunother. 29(6):616-27); Gp37(human leukemia T-cell antigen; Bhattacharya-Chatterjee et al. (1988)“Idiotype Vaccines Against Human T Cell Leukemia. IL Generation AndCharacterization Of A Monoclonal Idiotype Cascade (Ab1, Ab2, and Ab3),”J. Immunol. 141:1398-1403); gp75 (melanoma antigen; Vijayasardahl et al.(1990) “The Melanoma Antigen Gp75 Is The Human Homologue Of The Mouse B(Brown) Locus Gene Product,” J. Exp. Med. 171(4):1375-1380); gpA33(Heath, J. K. et al. (1997) “The Human A33 Antigen Is A TransmembraneGlycoprotein And A Novel Member Of The Immunoglobulin Superfamily,”Proc. Natl. Acad. Sci. (U.S.A.) 94(2):469-474; Ritter, G. et al. (1997)“Characterization Of Posttranslational Modifications Of Human A33Antigen, A Novel Palmitoylated Surface Glycoprotein Of HumanGastrointestinal Epithelium,” Biochem. Biophys. Res. Commun.236(3):682-686; Wong, N. A. et al. (2006) “EpCAM and gpA33 Are MarkersOf Barrett's Metaplasia,” J. Clin. Pathol. 59(3):260-263; Almqvist, Y.(2006) “In vitro and in vivo Characterization of 177Lu-huA33: ARadioimmunoconjugate Against Colorectal Cancer,” Nucl. Med. Biol.33(8):991-998); HER2 antigen (HER2/neu, p185^(HER2); Pal, S. K. et al.(2006) “Targeting HER2 Epitopes,” Semin. Oncol. 33(4):386-391); HMFG(human milk fat globule antigen; WO1995015171); HumanPapillomavirus-E6/Human Papillomavirus-E7 (DiMaio, D. et al. (2006)“Human Papillomaviruses And Cervical Cancer,” Adv. Virus Res. 66:125-59;HMW-MAA (high molecular weight melanoma antigen; Natali et al. (1987)“Immunohistochemical Detection Of Antigen In Human Primary AndMetastatic Melanomas By The Monoclonal Antibody 140.240 And Its PossiblePrognostic Significance,” Cancer 59:55-63; Mittelman et al. (1990)“Active Specific Immunotherapy In Patients With Melanoma. A ClinicalTrial With Mouse Antiidiotypic Monoclonal Antibodies Elicited WithSyngeneic Anti-High-Molecular-Weight-Melanoma-Associated AntigenMonoclonal Antibodies,” J. Clin. Invest. 86:2136-2144); I antigen(differentiation antigen; Feizi (1985) “Demonstration By MonoclonalAntibodies That Carbohydrate Structures Of Glycoproteins And GlycolipidsAre Onco-Developmental Antigens,” Nature 314:53-57); IL13Rα2 (PCTPublication No. WO 2008/146911; Brown, C. E. et al. (2013) “GliomaIL13Rα2 Is Associated With Mesenchymal Signature Gene Expression AndPoor Patient Prognosis,” PLoS One. 18; 8(10):e77769; Barderas, R. et al.(2012) “High Expression Of IL-13 Receptor A2 In Colorectal Cancer IsAssociated With Invasion, Liver Metastasis, And Poor Prognosis,” CancerRes. 72(11):2780-2790; Kasaian, M. T. et al. (2011) “IL-13 AntibodiesInfluence IL-13 Clearance In Humans By Modulating Scavenger Activity OfIL-13Rα2,” J. Immunol. 187(1):561-569; Bozinov, O. et al. (2010)“Decreasing Expression Of The Interleukin-13 Receptor IL-13Ralpha2 InTreated Recurrent Malignant Gliomas,” Neurol. Med. Chir. (Tokyo)50(8):617-621; Fujisawa, T. et al. (2009) “A novel role ofinterleukin-13 receptor alpha2 in pancreatic cancer invasion andmetastasis,” Cancer Res. 69(22):8678-8685); Integrin β6 (PCT PublicationNo. WO 03/087340); JAM-3 (PCT Publication No. WO 06/084078); KID3 (PCTPublication No. WO 05/028498); KID31 (PCT Publication No. WO 06/076584);KS 1/4 pan-carcinoma antigen (Perez et al. (1989) “Isolation AndCharacterization Of A cDNA Encoding The Ks1/4 Epithelial CarcinomaMarker,” J. Immunol. 142:3662-3667; Möller et al. (1991)“Bi-specific-Monoclonal-Antibody-Directed Lysis Of Ovarian CarcinomaCells By Activated Human T Lymphocytes,” Cancer Immunol. Immunother.33(4):210-216; Ragupathi, G. 2005 Cancer Treat Res. 123:157-80); L6 andL20 (human lung carcinoma antigens; Hellström et al. (1986) “MonoclonalMouse Antibodies Raised Against Human Lung Carcinoma,” Cancer Res.46:3917-3923); LEA; LUCA-2 (United States Patent Publication No.2006/0172349; PCT Publication No. WO 06/083852); M1:22:25:8; M18; M39;MAGE (MAGE-1; MAGE-3; (Bodey, B. (2002) “Cancer-Testis Antigens:Promising Targets For Antigen Directed Antineoplastic Immunotherapy,”Expert Opin. Biol. Ther. 2(6):577-584); MART (Kounalakis, N. et al.(2005) “Tumor Cell And Circulating Markers In Melanoma: Diagnosis,Prognosis, And Management,” Curr. Oncol. Rep. 7(5):377-382; mesothelin(Chang, K. et al. (1996) “Molecular Cloning Of Mesothelin, ADifferentiation Antigen Present On Mesothelium, Mesotheliomas, AndOvarian Cancers,” Proc. Natl. Acad. Sci. (U.S.A.) 93:136-140); MUC-1(Mathelin, C. (2006) “Circulating Proteinic Biomarkers And BreastCancer,” Gynecol. Obstet. Fertil. 34(7-8):638-646); MUM-1 (Castelli, C.et al. (2000) “T-Cell Recognition Of Melanoma-Associated Antigens,” J.Cell. Physiol. 182(3):323-331); Myl; N-acetylglucosaminyltransferase(Dennis, J. W. (1999) “Glycoprotein Glycosylation And CancerProgression,” Biochim. Biophys. Acta. 6; 1473(1):21-34);neoglycoprotein; NS-10 as found in adenocarcinomas; OFA-1; OFA-2;Oncostatin M (Oncostatin Receptor Beta; U.S. Pat. No. 7,572,896; PCTPublication No. WO 06/084092); p15 (Gil, J. et al. (2006) “Regulation OfThe INK4b-ARF-INK4a Tumour Suppressor Locus: All For One Or One ForAll,” Nat. Rev. Mol. Cell Biol. 7(9):667-677); p97 (melanoma-associatedantigen; Estin et al. (1989) “Transfected Mouse Melanoma Lines ThatExpress Various Levels Of Human Melanoma-Associated Antigen p97,” J.Natl. Cancer Instit. 81(6):445-454); PEM (polymorphic epithelial mucin;Hilkens et al. (1992) “Cell Membrane-Associated Mucins And TheirAdhesion-Modulating Property,” Trends in Biochem. Sci. 17:359-363); PEMA(polymorphic epithelial mucin antigen); PIPA (U.S. Pat. No. 7,405,061;PCT Publication No. WO 04/043239); PSA (prostate-specific antigen;Henttu et al. (1989) “cDNA Coding For The Entire Human Prostate SpecificAntigen Shows High Homologies To The Human Tissue Kallikrein Genes,”Biochem. Biophys. Res. Comm. 10(2):903-910; Israeli et al. (1993)“Molecular Cloning Of A Complementary DNA Encoding A Prostate-SpecificMembrane Antigen,” Cancer Res. 53:227-230; Cracco, C. M. et al. (2005)“Immune Response In Prostate Cancer,” Minerva Urol. Nefrol.57(4):301-311); PSMA (prostate-specific membrane antigen; Ragupathi, G.(2005) “Antibody Inducing Polyvalent Cancer Vaccines,” Cancer Treat.Res. 123:157-180); prostatic acid phosphate (Tailor et al. (1990)“Nucleotide Sequence Of Human Prostatic Acid Phosphatase Determined FromA Full-Length cDNA Clone,” Nucl. Acids Res. 18(16):4928); R₂₄ as foundin melanoma; ROR1 (U.S. Pat. No. 5,843,749); sphingolipids; SSEA-1;SSEA-3; SSEA-4; sTn (Holmberg, L. A. (2001) “Theratope Vaccine(STn-KLH),” Expert Opin. Biol. Ther. 1(5):881-91); T-cell receptorderived peptide from a cutaneous T-cell lymphoma (see Edelson (1998)“Cutaneous T-Cell Lymphoma: A Model For Selective Immunotherapy,” CancerJ. Sci. Am. 4:62-71); T₅A₇ found in myeloid cells; TAG-72 (Yokota et al.(1992) “Rapid Tumor Penetration Of A Single-Chain Fv And Comparison WithOther Immunoglobulin Forms,” Cancer Res. 52:3402-3408); TL5 (blood groupA); TNF-receptor (TNF-α receptor, TNF-ß receptor; TNF-γ receptor (vanHorssen, R. et al. (2006) “TNF-Alpha In Cancer Treatment: MolecularInsights, Antitumor Effects, And Clinical Utility,” Oncologist11(4):397-408; Gardnerova, M. et al. (2000) “The Use Of TNF FamilyLigands And Receptors And Agents Which Modify Their Interaction AsTherapeutic Agents,” Curr. Drug Targets 1(4):327-364); TRA-1-85 (bloodgroup H); Transferrin Receptor (U.S. Pat. No. 7,572,895; PCT PublicationNo. WO 05/121179); 5T4 (TPBG, trophoblast glycoprotein; Boghaert, E. R.et al. (2008) “The Oncofetal Protein, 5T4, Is A Suitable Target ForAntibody-Guided Anti-Cancer Chemotherapy With Calicheamicin,” Int. J.Oncol. 32(1):221-234; Eisen, T. et al. (2014) “Naptumomab Estafenatox:Targeted Immunotherapy with a Novel Immunotoxin,” Curr. Oncol. Rep.16:370, pp. 1-6); TSTA (tumor-specific transplantation antigen) such asvirally-induced tumor antigens including T-antigen DNA tumor viruses andenvelope antigens of RNA tumor viruses, oncofetalantigen-alpha-fetoprotein such as CEA of colon, bladder tumor oncofetalantigen (Hellström et al. (1985) “Monoclonal Antibodies To Cell SurfaceAntigens Shared By Chemically Induced Mouse Bladder Carcinomas,” Cancer.Res. 45:2210-2188); VEGF (Pietrantonio, F. et al. (2015)“Bevacizumab-Based Neoadjuvant Chemotherapy For Colorectal Cancer LiverMetastases: Pitfalls And Helpful Tricks In A Review For Clinicians,”Crit. Rev. Oncol. Hematol. 95(3):272-281; Grabowski, J. P. (2015)“Current Management Of Ovarian Cancer,” Minerva Med. 106(3):151-156;Field, K. M. (2015) “Bevacizumab And Glioblastoma: Scientific Review,Newly Reported Updates, And Ongoing Controversies,” Cancer121(7):997-1007; Suh, D. H. et al. (2015) “Major Clinical ResearchAdvances In Gynecologic Cancer In 2014,” J. Gynecol. Oncol.26(2):156-167; Liu, K. J. et al. (2015) “Bevacizumab In Combination WithAnticancer Drugs For Previously Treated Advanced Non-Small Cell LungCancer,” Tumour Biol. 36(3):1323-1327; Di Bartolomeo, M. et al. (2015)“Bevacizumab Treatment In The Elderly Patient With Metastatic ColorectalCancer,” Clin. Interv. Aging 10:127-133); VEGF Receptor (O'Dwyer. P. J.(2006) “The Present And Future Of Angiogenesis-Directed Treatments OfColorectal Cancer,” Oncologist 11(9):992-998); VEP8; VEP9; VIM-D5; and Yhapten, Le^(y) as found in embryonal carcinoma cells. Additional CancerAntigens, and molecules (e.g., antibodies) that bind them are disclosedin Table 7. 5T4, B7-H3, CEACAM5/CEACAM6, CD123, DR5, EGFR, an Ephrinreceptor, gpA33, HER2/neu, IL13Rα2, ROR1, and VEGF are particularlypreferred “Cancer Antigens” of the present invention.

TABLE 7 Antibody and Antibody-Based Molecules Antibody Name CancerAntigens Therapeutic Target Application 3F8 Gd2 Neuroblastoma 8H9 B7-H3Neuroblastoma, Sarcoma, Metastatic Brain Cancers Abagovomab CA-125Ovarian Cancer Adecatumumab Epcam Prostate and Breast Cancer AfutuzumabCD20 Lymphoma Alacizumab VEGFR2 Cancer Altumomab CEA Colorectal CancerAmatuximab Mesothelin Cancer Anatumomab TAG-72 Non-Small Cell LungCarcinoma Mafenatox Anifrolumab Interferon A/B Systemic LupusErythematosus Receptor Anrukinzumab IL-13 Cancer Apolizumab HLA-DRHematological Cancers Arcitumomab CEA Gastrointestinal Cancer AtinumabRTN4 Cancer Bectumomab CD22 Non-Hodgkin's Lymphoma (Detection) BelimumabBAFF Non-Hodgkin Lymphoma Bevacizumab VEGF-A Metastatic Cancer,Retinopathy of Prematurity Bivatuzumab CD44 V6 Squamous Cell CarcinomaBlinatumomab CD19 Cancer Brentuximab CD30 (TNFRSF8) Hematologic CancersCantuzumab MUC1 Cancers Cantuzumab Mucin Canag Colorectal CancerMertansine Caplacizumab VWF Cancers Capromab Prostatic CarcinomaProstate Cancer (Detection) Cells Carlumab MCP-1 Oncology/ImmuneIndications Catumaxomab Epcam, CD3 Ovarian Cancer, Malignant Ascites,Gastric Cancer Cc49 Tag-72 Tumor Detection Cetuximab EGFR MetastaticColorectal Cancer and Head and Neck Cancer Ch.14.18 UndeterminedNeuroblastoma Citatuzumab Epcam Ovarian Cancer and other Solid TumorsCixutumumab IGF-1 Receptor Solid Tumors Clivatuzumab MUC1 PancreaticCancer Conatumumab TRAIL-R2 Cancer Dacetuzumab CD40 Hematologic CancersDalotuzumab Insulin-Like Growth Cancer Factor I Receptor DaratumumabCD38 Cancer Demcizumab DLL4 Cancer Detumomab B-Lymphoma Cell LymphomaDrozitumab DR5 Cancer Duligotumab HER3 Cancer Dusigitumab ILGF2 CancerEcromeximab GD3 Ganglioside Malignant Melanoma Eculizumab C5 ParoxysmalNocturnal Hemoglobinuria Edrecolomab Epcam Colorectal CarcinomaElotuzumab SLAMF7 Multiple Myeloma Elsilimomab IL-6 Cancer EnavatuzumabTWEAK Receptor Cancer Enlimomab ICAM-1 (CD54) Cancer Enokizumab IL9Asthma Enoticumab DLL4 Cancer Ensituximab 5AC Cancer EpitumomabEpisialin Cancer Cituxetan Epratuzumab CD22 Cancer, SLE ErtumaxomabHER2/Neu, CD3 Breast Cancer Etaracizumab Integrin A_(v)β₃ Melanoma,Prostate Cancer, Ovarian Cancer Faralimomab Interferon Receptor CancerFarletuzumab Folate Receptor 1 Ovarian Cancer Fasinumab HNGF CancerFbta05 CD20 Chronic Lymphocytic Leukaemia Ficlatuzumab HGF CancerFigitumumab IGF-1 Receptor Adrenocortical Carcinoma, Non-Small Cell LungCarcinoma Flanvotumab TYRP1 Melanoma (Glycoprotein 75) FontolizumabIFN-γ Crohn's Disease Fresolimumab TGF-B Idiopathic Pulmonary Fibrosis,Focal Segmental Glomerulosclerosis, Cancer Futuximab EGFR CancerGaliximab CD80 B Cell Lymphoma Ganitumab IGF-I Cancer Gemtuzumab CD33Acute Myelogenous Leukemia Ozogamicin Gevokizumab IL-1β DiabetesGirentuximab Carbonic Anhydrase Clear Cell Renal Cell Carcinoma 9(CA-IX) Glembatumumab GPNMB Melanoma, Breast Cancer Vedotin GolimumabTNF-A Rheumatoid Arthritis, Psoriatic Arthritis, Ankylosing SpondylitisIbritumomab CD20 Non-Hodgkin's Lymphoma Tiuxetan Icrucumab VEGFR-1Cancer Igovomab CA-125 Ovarian Cancer (Diagnosis) Imab362 Cldn18.2Gastrointestinal Adenocarcinomas and Pancreatic Tumor Imgatuzumab EGFRCancer Inclacumab Selectin P Cancer Indatuximab SDC1 Cancer RavtansineInotuzumab CD22 Cancer Ozogamicin Intetumumab CD51 Solid Tumors(Prostate Cancer, Melanoma) Ipilimumab CD152 Melanoma Iratumumab CD30(TNFRSF8) Hodgkin's Lymphoma Itolizumab CD6 Cancer Labetuzumab CEAColorectal Cancer Lambrolizumab PDCD1 Antineoplastic Agent LampalizumabCFD Cancer Lexatumumab TRAIL-R2 Cancer Libivirumab Hepatitis B SurfaceHepatitis B Antigen Ligelizumab IGHE Cancer Lintuzumab CD33 CancerLirilumab KIR2D Cancer Lorvotuzumab CD56 Cancer Lucatumumab CD40Multiple Myeloma, Non-Hodgkin's Lymphoma, Hodgkin's Lymphoma LumiliximabCD23 Chronic Lymphocytic Leukemia Mapatumumab TRAIL-R1 CancerMargetuximab Ch4d5 Cancer Matuzumab EGFR Colorectal, Lung and StomachCancer Milatuzumab CD74 Multiple Myeloma and Other HematologicalMalignancies Minretumomab TAG-72 Cancer Mitumomab GD3 Ganglioside SmallCell Lung Carcinoma Mogamulizumab CCR4 Cancer Morolimumab Rhesus FactorCancer Moxetumomab CD22 Cancer Pasudotox Nacolomab C242 AntigenColorectal Cancer Tafenatox Namilumab CSF2 Cancer Naptumomab 5T4Non-Small Cell Lung Estafenatox Carcinoma, Renal Cell CarcinomaNarnatumab RON Cancer Nebacumab Endotoxin Sepsis Necitumumab EGFRNon-Small Cell Lung Carcinoma Nerelimomab TNF-A Cancer NesvacumabAngiopoietin 2 Cancer Nimotuzumab EGFR Squamous Cell Carcinoma, Head andNeck Cancer, Nasopharyngeal Cancer, Glioma Nivolumab PD-1 CancerNofetumomab Undetermined Cancer Merpentan Ocaratuzumab CD20 CancerOfatumumab CD20 Chronic Lymphocytic Leukemia Olaratumab PDGF-R A CancerOlokizumab IL6 Cancer Onartuzumab Human Scatter Cancer Factor ReceptorKinase Ontuxizumab TEM1 Cancer Oportuzumab Epcam Cancer MonatoxOregovomab CA-125 Ovarian Cancer Orticumab Oxldl Cancer OtlertuzumabCD37 Cancer Panitumumab EGFR Colorectal Cancer Pankomab Tumor SpecificOvarian Cancer Glycosylation of MUC1 Parsatuzumab EGFL7 CancerPatritumab HER3 Cancer Pembrolizumab PD-1 Cancer Pemtumomab MUC1 CancerPerakizumab IL17A Arthritis Pertuzumab HER2/Neu Cancer Pidilizumab PD-1Cancer and Infectious Diseases Pinatuzumab CD22 Cancer VedotinPintumomab Adenocarcinoma Adenocarcinoma Antigen Placulumab Human TNFCancer Polatuzumab CD79B Cancer Vedotin Pritoxaximab E. Coli Shiga ToxinCancer Type-1 Pritumumab Vimentin Brain Cancer Quilizumab IGHE CancerRacotumomab N- Cancer Glycolylneuraminic Acid Radretumab FibronectinExtra Cancer Domain-B Ramucirumab VEGFR2 Solid Tumors Rilotumumab HGFSolid Tumors Rituximab CD20 Lymphomas, Leukemias, Some AutoimmuneDisorders Robatumumab IGF-1 Receptor Cancer Roledumab RHD CancerSamalizumab CD200 Cancer Satumomab TAG-72 Cancer Pendetide SeribantumabERBB3 Cancer Setoxaximab E. Coli Shiga Toxin Cancer Type-1 Sgn-CD19aCD19 Acute Lymphoblastic Leukemia and B Cell Non-Hodgkin LymphomaSgn-CD33a CD33 Acute Myeloid Leukemia Sibrotuzumab FAP Cancer SiltuximabIL-6 Cancer Solitomab Epcam Cancer Sontuzumab Episialin Cancer TabalumabBAFF B Cell Cancers Tacatuzumab Alpha-Fetoprotein Cancer TetraxetanTaplitumomab CD19 Cancer Paptox Telimomab Undetermined CancerTenatumomab Tenascin C Cancer Teneliximab CD40 Cancer Teprotumumab CD221Hematologic Tumors Ticilimumab CTLA-4 Cancer Tigatuzumab TRAIL-R2 CancerTnx-650 Il-13 Hodgkin's Lymphoma Tositumomab CD20 Follicular LymphomaTovetumab CD140a Cancer Trastuzumab HER2/Neu Breast Cancer Trbs07 Gd2Melanoma Tremelimumab CTLA-4 Cancer Tucotuzumab Epcam Cancer CelmoleukinUblituximab MS4A1 Cancer Urelumab 4-1BB Cancer Vantictumab FrizzledReceptor Cancer Vapaliximab AOC3 (VAP-1) Cancer Vatelizumab ITGA2 CancerVeltuzumab CD20 Non-Hodgkin's Lymphoma Vesencumab NRP1 CancerVolociximab Integrin A5β1 Solid Tumors Vorsetuzumab CD70 CancerVotumumab Tumor Antigen Colorectal Tumors CTAA16.88 Zalutumumab EGFRSquamous Cell Carcinoma of The Head And Neck Zatuximab HER1 CancerZiralimumab CD147 Cancer

Exemplary antibodies, whose VH and VL Domains may be used to constructthe Binding Molecules of the present invention that are capable ofbinding a Cancer Antigen arrayed on the surface of a cancer cell andmediating the redirected killing of such cancer cells are listed inTable 7 above, additional antibodies that may be used to constructmolecules capable of binding a Cancer Antigen arrayed on the surface ofa cancer cell and mediating the redirected killing of such cancer cellsare provided below.

1. Exemplary Anti-B7-H3 Antibodies

B7-H3 is a Cancer Antigen that is overexpressed on a wide variety ofsolid tumor types and is a member of the B7 family of molecules that areinvolved in immune regulation (see, U.S. Pat. No. 8,802,091; US2014/0328750; US 2013/0149236; Loo, D. et al. (2012) “Development Of AnFc-Enhanced Anti-B7-H3 Monoclonal Antibody With Potent AntitumorActivity,” Clin. Cancer Res. 18(14):3834-3845). In particular, severalindependent studies have shown that human malignant cancer cells (e.g.,cancer cells of neuroblastomas and gastric, ovarian, pancreatic, andnon-small cell lung cancers) exhibit a marked increase in expression ofB7-H3 protein and that this increased expression was associated withincreased disease severity (Zang, X. et al. (2007) “The B7 Family AndCancer Therapy: Costimulation And Coinhibition,” Clin. Cancer Res.13:5271-5279), suggesting that B7-H3 is exploited by tumors as an immuneevasion pathway (Hofmeyer, K. et al. (2008) “The Contrasting Role OfB7-H3,” Proc. Natl. Acad. Sci. (U.S.A.) 105(30):10277-10278).

B7-H3 has also been found to co-stimulate CD4+ and CD8+ T-cellproliferation. B7-H3 also stimulates IFN-7 production and CD8⁺ lyticactivity (Chapoval, A. et al. (2001) “B7-H3: A Costimulatory MoleculeFor T Cell Activation and IFN-γ Production,” Nature Immunol. 2:269-274;Sharpe, A. H. et al. (2002) “The B7-CD28 Superfamily,” Nature Rev.Immunol. 2:116-126). However, the protein also possibly acts throughNFAT (nuclear factor for activated T-cells), NF-κB (nuclear factor kappaB), and AP-1 (Activator Protein-1) factors to inhibit T-cell activation(Yi. K. H. et al. (2009) “Fine Tuning The Immune Response Through B7-H3And B7-H4,” Immunol. Rev. 229:145-151). B7-H3 is also believed toinhibit Th1, Th2, or Th17 in vivo (Prasad, D. V. et al. (2004) “MurineB7-H3 Is A Negative Regulator Of T Cells,” J. Immunol. 173:2500-2506;Fukushima, A. et al. (2007) “B7-H3 Regulates The Development OfExperimental Allergic Conjunctivitis In Mice,” Immunol. Lett. 113:52-57;Yi. K. H. et al. (2009) “Fine Tuning The Immune Response Through B7-H3And B7-H4,” Immunol. Rev. 229:145-151).

Preferred B7-H3-Binding Molecules possess the VL and/or VH Domains, ofhumanized anti-human B7-H3 monoclonal antibody “B7-H3 mAb-B,” “B7-H3mAb-C,” “B7-H3 mAb-D,” or any of the anti-B7-H3 antibodies providedherein; and more preferably possess 1, 2 or all 3 of the CDR_(L)s of theVL Domain and/or 1, 2 or all 3 of the CDR_(H)s of the VH Domain of suchanti-B7-H3 monoclonal antibodies.

Upon humanization, antibody B7-H3 mAb-B yielded two variant VH Domains,B7-H3 mAb-B VH1 and B7-H3 mAb-B VH2; and two variant VL Domains B7-H3mAb-B VH1 VL1 and B7-H3 mAb-B VL2, which may be used in any combinationof VH/VL Domains to yield a functional B7-H3 Binding Domain.

The amino acid sequence of the VH Domain of B7-H3 mAb-B VH1 is SEQ IDNO:122 (CDR_(H) residues are shown underlined):

QVQLVQSGAE VKKPGASVKV SCKASGYTFT  SYWMQ WVRQA PGQGLEWMG T   IYPGDGDTRY  TQKFKG RVTI TADKSTSTAY MELSSLRSED TAVYYCAR RG   IPRLWYFDV W GQGTTVTVSS

The amino acid sequence of the VH Domain of B7-H3 mAb-B VH2 is SEQ IDNO:123 (CDR_(H) residues are shown underlined):

QVQLVQSGAE VKKPGASVKV SCKASGYTFT  SYWMQ WVRQA PGQGLEWMG T   IYPGGGDTRY  TQKFQG RVTI TADKSTSTAY MELSSLRSED TAVYYCAR RG   IPRLWYFDV W GQGTTVTVSS

The amino acid sequence of the VL Domain of B7-H3 mAb-B VL1 is SEQ IDNO:124 (CDR_(L) residues are shown underlined).

DIQMTQSPSS LSASVGDRVT ITC RASQDIS   NYLN WYQQKP GKAPKLLIY Y   TSRLHSGVPS RFSGSGSGTD FTLTISSLQP EDIATYYC QQ   GNTLPPT FGG GTKLEIK

The amino acid sequence of the VL Domain of B7-H3 mAb-B VL2 is SEQ IDNO:125 (CDR_(L) residues are shown underlined).

DIQMTQSPSS LSASVGDRVT ITC RASQSIS   SYLN WYQQKP GKAPKLLIY Y   TSRLQSGVPS RFSGSGSGTD FTLTISSLQP EDIATYYC QQ   GNTLPPT FCG GTKLEIK

The amino acid sequence of the VH Domain of humanized B7-H3 mAb-C is SEQID NO:126 (CDR_(H) residues are shown underlined):

EVQLVESGGG LVKPGGSLRL SCAASGFTFS  SYGMS WVRQA PGKGLEWVA T  INSGGSNTYY PDSLKG RFTI SRDNAKNSLY LQMNSLRAED TAVYYCAR HD   GGAMDYWGQG TTVTVSS

The amino acid sequence of the VL Domain of humanized B7-H3 mAb-C is SEQID NO:127 (CDR_(L) residues are shown underlined).

DIQMTQSPSS LSASVGDRVT ITC RASESIY SYLA WYQQKP GKAPKLLVY N   TKTLPEGVPS RFSGSGSGTD FTLTISSLQP EDFATYYC QH HYGTPPWT FG QGTRLEIK

The amino acid sequence of the VH Domain of B7-H3 mAb-D (SEQ ID NO:128)is shown below (CDR_(H) residues are shown underlined).

EVQLVESGGG LVQPGGSLRL SCAASGFTFS  SFGMH WVRQAPGKGLEWVAY ISSGSGTIYY ADTVKGRFTI SRDNAKNSLY LQMNSLRAED TAVYYCAR HG  YRYEGFDY WG QGTTVTVSS

The amino acid sequence of the VL Domain of B7-H3 mAb-D (SEQ ID NO:129)is shown below (CDR_(L) residues are shown underlined).

DIQMTQSPSF LSASVGDRVT ITC KASQNVD TNVA WYQQKP GKAPKALIY S   ASYRYSGVPS RFSGSGSGTD FTLTISSLQP EDFAEYFC QQ YNNYPFT FGQ GTKLEIK

Particularly preferred, are B7-H3-Binding Molecules which possess ahumanized VH and/or VL Domain including but not limited to“Enoblituzumab” (also known as MGA271; CAS Reg No. 1353485-38-7).Enoblituzumab is an Fc-optimized monoclonal antibody that binds toHER2/neu and mediates enhanced ADCC activity. The amino acid sequencesof the complete Heavy and Light Chains of Enoblituzumab are known in theart (see., e.g., WHO Drug Information, 2017, Recommended INN: List 77,31(1):49). The amino acid sequence of the VH Domain of Enoblituzumab is(SEQ ID NO:130) (CDR_(H)s are underlined):

EVQLVESGGG LVQPGGSLRL SCAASGFTFS  SFGMH WVRQA PGKGLEWVA Y  ISSDSSAIYY ADTVKG RFTI SRDNAKNSLY LQMNSLRDED TAVYYCGR GR   ENIYYGSRLD YWGQGTTVTV SSThe amino acid sequence of the VL Domain of Enoblituzumab is (SEQ IDNO:131) (CDR_(L)s are underlined):

DIQLTQSPSF LSASVGDRVT ITC KASQNVD TNVA WYQQKP GKAPKALIY S   ASYRYSGVPS RFSGSGSGTD FTLTISSLQP EDFATYYC QQ YNNYPFT FGQ GTKLEIK

In addition to the above-identified preferred anti-B7-H3 BindingMolecules, the invention contemplates the use of any of the followinganti-B7-H3 Binding Molecules: LUCA1; BLA8; PA20; or SKN2 (see, U.S. Pat.Nos. 7,527,969; 8,779,098 and PCT Patent Publication WO 2004/001381);M30; cM30; M30-H1-L1; M30-H1-L2; M30-H1-L3; M30-H1-L4; M30-H1-L5;M30-H1-L6; M30-H1-L7; M30-H4-L1; M30-H4-L2; M30-H4-L3; and M30-H4-L4(see, US Patent Publication 2013/0078234 and PCT Patent Publication WO2012/147713); and 8H9 (see U.S. Pat. Nos. 7,666,424; 7,737,258;7,740,845; 8,148,154; 8,414,892; 8,501,471; 9,062,110; US PatentPublication 2010/0143245 and PCT Patent Publication WO 2008/116219).

2. Exemplary Anti-CEACAM5 and Anti-CEACAM6 Antibodies

Carcinoembryonic Antigen-Related Cell Adhesion Molecules 5 (CEACAM5) and6 (CEACAM6) have been found to be associated with various types ofcancers including medullary thyroid cancer, colorectal cancer,pancreatic cancer, hepatocellular carcinoma, gastric cancer, lungcancer, head and neck cancers, urinary bladder cancer, prostate cancer,uterine cancer, endometrial cancer, breast cancer, hematopoietic cancer,leukemia and ovarian cancer (PCT Publication No. WO 2011/034660), andparticularly colorectal, gastrointestinal, pancreatic, non-small celllung cancer (NSCL), breast, thyroid, stomach, ovarian and uterinecarcinomas (Zheng, C. et al. (2011) “A Novel Anti-CEACAM5 MonoclonalAntibody, CC4, Suppresses Colorectal Tumor Growth and Enhances NKCells-Mediated Tumor Immunity,” PLoS One 6(6):e21146, pp. 1-11).

CEACAM5 has been found to be overexpressed in 90% of gastrointestinal,colorectal and pancreatic cancers, 70% of non-small cell lung cancercells and 50% of breast cancers (Thompson, J. A. et al. (1991)“Carcinoembryonic Antigen Gene Family: Molecular Biology And ClinicalPerspectives,” J. Clin. Lab. Anal. 5:344-366). Overexpressedcarcinoembryonic antigen-related cellular adhesion molecule 6 (CEACAM6)plays important roles in the invasion and metastasis of a variety ofhuman cancers, including medullary thyroid cancer, colorectal cancer,pancreatic cancer, hepatocellular carcinoma, gastric cancer, lungcancer, head and neck cancers, urinary bladder cancer, prostate cancer,uterine cancer, endometrial cancer, breast cancer, hematopoietic cancer,leukemia and ovarian cancer (PCT Publication No. WO 2011/034660; Deng,X. et al. (2014) “Expression Profiling Of CEACAM6 Associated With TheTumorigenesis And Progression In Gastric Adenocarcinoma,” Genet. Mol.Res. 13(3):7686-7697; Cameron, S. et al. (2012) “Focal Overexpression OfCEACAM6 Contributes To Enhanced Tumourigenesis In Head And Neck CancerVia Suppression Of Apoptosis,” Mol. Cancer 11:74, pp. 1-11; Chapin, C.et al. (2012) “Distribution And Surfactant Association OfCarcinoembryonic Cell Adhesion Molecule 6In Human Lung,” Amer. J.Physiol. Lung Cell. Mol. Physiol. 302(2):L216-L25; Riley, C. J. et al.(2009) “Design And Activity Of A Murine And Humanized Anti-CEACAM6Single-Chain Variable Fragment In The Treatment Of Pancreatic Cancer,”Cancer Res. 69(5):1933-1940; Lewis-Wambi, J. S. et al. (2008)“Overexpression Of CEACAM6 Promotes Migration And Invasion OfOestrogen-Deprived Breast Cancer Cells,” Eur. J. Cancer44(12):1770-1779; Blumenthal, R. D. et al. (2007) “Expression PatternsOf CEACAM5 And CEACAM6 In Primary And Metastatic Cancers,” BMC Cancer.7:2, pp. 1-15). Antibodies that immunospecifically bind CEACAM5 andCEACAM6 are commercially available (Santa Cruz Biotechnology, Inc.,Novus Biologicals LLC; Abnova Corporation).

The amino acid sequence of the VH Domain of the humanizedanti-CEACAM5/ANTI-CEACAM6 antibody 16C3 (EP 2585476) (SEQ ID NO:132) isshown below (CDR_(H) residues are shown underlined):

QVQLQQSGPE VVRPGVSVKI SCKGS GYTFT DYAMH WVKQS MAKSLEWIG L  ISTYSGDTKY NQNFKG KATM TVDKSASTAY MELSSLRSED TAVYYCAR GD   YSGSRYWFAY WGQGTLVTVS S

The amino acid sequence of the VL Domain of the humanizedanti-CEACAM5/ANTI-CEACAM6 antibody 16C3 (EP 2585476) (SEQ ID NO:133) isshown below (CDR_(L) residues are shown underlined):

DIQMTQSPSS LSASVGDRVT ITC GASENIY GALN WYQRKP GKSPKLLIW G   ASNLADGMPS RFSGSGSGRQ YTLTISSLQP EDVATYY CQN VLSSPYT FGG GTKLEIK

The amino acid sequence of the VH Domain of the humanizedanti-CEACAM5/CEACAM6 antibody hMN15 (WO 2011/034660) (SEQ ID NO:134) isshown below (CDR_(H) residues are shown underlined):

QVQLVESGGG VVQPGRSLRL SC SSSGFALT DYYMS WVRQA PGKGLEWLG F  IANKANGHTT DYSPSVKG RF TISRDNSKNT LFLQMDSLRP EDTGVYFCAR  DMGIRWNFDV WGQGTPVTVS S

The amino acid sequence of the VL Domain of the humanizedanti-CEACAM5/CEACAM6 antibody hMN15 (WO 2011/034660) (SEQ ID NO:135) isshown below (CDR_(L) residues are shown underlined):

DIQLTQSPSS LSASVGDRVT MTC SASSRVS YIH WYQQKPG KAPKRWIY GT   STLASGVPAR FSGSGSGTDF TFTISSLQPE DIATYYC QQW SYNPPT FGQG TKVEIKR

The present invention specifically includes and encompassesCEACAM5/CEACAM6 Binding Molecules (e.g., CEACAM5/CEACAM6×CD3 bispecificBinding Molecules) that are capable of binding to CEACAM5 and/orCEACAM6, and particularly such Binding Molecules that comprise the VLand/or VH Domain, and/or 1, 2 or all 3 of the CDR_(L)s of the VL Domainand/or 1, 2 or all 3 of the CDR_(H)s of the VH Domain of theanti-CEACAM5/CEACAM6 monoclonal antibodies 16C3 or hMN15.

3. Exemplary Anti-EGRF Antibodies

Epidermal Growth Factor Receptor (EGFR) is a Cancer Antigen of certainmetastatic colorectal cancer, metastatic non-small cell lung cancer andhead and neck cancer. Exemplary antibodies that bind human EGRF are“Cetuximab” and “Panitumumab.” Cetuximab is a recombinant human-mousechimeric epidermal growth factor receptor (EGFR) IgG1 monoclonalantibody (Govindan R. (2004) “Cetuximab In Advanced Non-Small Cell LungCancer,” Clin. Cancer Res. 10(12 Pt 2):4241s-4244s; Bou-Assaly, W. etal. (2010) “Cetuximab (Erbitux),” Am. J. Neuroradiol. 31(4):626-627).Panitumumab (Vectibix®, Amgen) is a fully humanized epidermal growthfactor receptor (EGFR) IgG2 monoclonal antibody (Foon, K. A. et al.(2004) “Preclinical And Clinical Evaluations Of ABX-EGF, A Fully HumanAnti-Epidermal Growth Factor Receptor Antibody,” Int. J. Radiat. Oncol.Biol. Phys. 58(3):984-990; Yazdi, M. H. et al. (2015) “A ComprehensiveReview of Clinical Trials on EGFR Inhibitors Such as Cetuximab andPanitumumab as Monotherapy and in Combination for Treatment ofMetastatic Colorectal Cancer,” Avicenna J. Med. Biotechnol.7(4):134-144).

The amino acid sequence of the VH Domain of the chimeric anti-EGFRantibody Cetuximab (SEQ ID NO:136) is shown below (CDR_(H) residues areshown underlined):

QVQLKQSGPG LVQPSQSLSI TCTVS GFSLT   NYGVH WVRQS PGKGLEWLG V  IWSGGNTDYN TPFTS RLSIN KDNSKSQVFF KMNSLQSNDT AIYYCAR ALT   Y YDYEFAYWG QGTLVIVSA

The amino acid sequence of the VL Domain of the chimeric anti-EGFRantibody Cetuximab (SEQ ID NO:137) is shown below (CDR_(L) residues areshown underlined):

DILLTQSPVI LSVSPGERVS FSC RASQSIG TNIH WYQQRT NGSPRLLIK Y   ASESISGIPS RFSGSGSGTD FTLSINSVES EDIADYYC QQ NNNWPTT FGA GTKLELKR

The amino acid sequence of the VH Domain of Panitumumab (SEQ ID NO:138)is shown below (CDR_(H) residues are shown underlined):

QVQLQESGPG LVKPSETLSL TCTVS GGSVS   SGDYY WTWIR QSPGKGLEWI G HIYYSGNTN  YNPSLKS RLT ISIDTSKTQF SLKLSSVTAA DTAIYYCVR D   RVTGAFDI WG QGTMVTVSS

The amino acid sequence of the VL Domain of Panitumumab (SEQ ID NO:139)is shown below (CDR_(L) residues are shown underlined):

DIQMTQSPSS LSASVGDRVT ITC QASQDIS   NYLN WYQQKP GKAPKLLIY D   ASNLETGVPS RFSGSGSGTD FTFTISSLQP EDIATYFC QH   FDHLPLA FGG GTKVEIKR

The present application specifically includes and encompasses EGFRBinding Molecules (e.g., EGFR×CD3 bispecific Binding Molecules) that arecapable of binding to EGFR, and particularly such Binding Molecules thatcomprise the VL and/or VH Domain, and/or 1, 2 or all 3 of the CDR_(L)sof the VL Domain and/or 1, 2 or all 3 of the CDR_(H)s of the VH Domainof the anti-EGFR monoclonal antibodies Cetuximab or Panitumumab.

4. Exemplary Anti-EphA2 Antibodies

The receptor tyrosine kinase, Ephrin type-A receptor 2 (EphA2) isnormally expressed at sites of cell-to-cell contact in adult epithelialtissues, however, recent studies have shown that it is alsooverexpressed in various types of epithelial carcinomas, with thegreatest level of EphA2 expression observed in metastatic lesions. Highexpression levels of EphA2 have been found in a wide range of cancersand in numerous cancer cell lines, including prostate cancer, breastcancer, non-small cell lung cancer and melanoma (Xu, J. et al. (2014)“High EphA2 Protein Expression In Renal Cell Carcinoma Is AssociatedWith A Poor Disease Outcome,” Oncol. Lett. August 2014; 8(2): 687-692;Miao, B. et al. (2014) “EphA2 is a Mediator of Vemurafenib Resistanceand a Novel Therapeutic Target in Melanoma,” Cancer Discov. pii:CD-14-0295). EphA2 does not appear to be merely a marker for cancer, butrather appears to be persistently overexpressed and functionally changedin numerous human cancers (Chen, P. et al. (2014) “EphA2 Enhances TheProliferation And Invasion Ability Of LnCap Prostate Cancer Cells,”Oncol. Lett. 8(1):41-46). Exemplary antibodies that bind human EphA2 are“EphA2 mAb 1,” “EphA2 mAb 2” and “EphA2 mAb 3.”

The amino acid sequence of the VH Domain of EphA2 mAb 1 (SEQ ID NO:140)is shown below (CDR_(H) residues are shown underlined):

QVQLKESGPG LVAPSQSLSI TCTVSGFSLS  RYSVH WVRQP PGKGLEWLG M   IWGGGSTDYN SALKSRLSIS KDNSKSQVFL KMNSLQTDDT AMYYCAR KHG   NYYTMDY WGQ GTSVTVSS

The amino acid sequence of the VL Domain of EphA2 mAb 1 (SEQ ID NO:141)is shown below (CDR_(L) residues are shown underlined):

DIQMTQTTSS LSASLGDRIT ISC RASQDIS NYLN WYQQKP DGTVKLLIY Y   TSRLHSGVPS RFSGSGSGTD YSLTISNLEQ EDIATYFC QQ GYTLYT FGGG TKLEIK

The amino acid sequence of the VH Domain of EphA2 mAb 2 (SEQ ID NO:142)is shown below (CDR_(H) residues are shown underlined):

QIQLVQSGPE LKKPGETVKI SCKASGFTFT  NYGMN WVKQA PGKGLKWMG W  INTYIGEPTY ADDFKG RFVF SLETSASTAY LQINNLKNED MATYFCAR EL   GPYYFDYWGQ GTTLTVSS

The amino acid sequence of the VL Domain of EphA2 mAb 2 (SEQ ID NO:143)is shown below (CDR_(L) residues are shown underlined):

DVVMTQTPLS LPVSLGDQAS ISC RSSQSLV HSSGNTYLH W YLQKPGQSPK LLIY KVSNRF SGVPDRFSGS GSGTDFTLKI SRVEAEDLGV YFC SQSTHVP   T FGSGTKLEI K

The amino acid sequence of the VH Domain of EphA2 mAb 3 (SEQ ID NO:144)is shown below (CDR_(H) residues are shown underlined):

EVQLVESGGG SVKPGGSLKL SCAASGFTFT  DHYMY WVRQT PEKRLEWVA T  ISDGGSFTSY PDSVKG RFTI SRDIAKNNLY LQMSSLKSED TAMYYCTR DE   SDRPFPYWGQ GTLVTVSS

The amino acid sequence of the VL Domain of EphA2 mAb 3 (SEQ ID NO:145)is shown below (CDR_(L) residues are shown underlined):

DIVLTQSHRS MSTSVGDRVN ITC KASQDVT TAVA WYQQKP GQSPKLLIF W   ASTRHAGVPD RFTGSGSGTD FTLTISSVQA GDLALYYC QQ HYSTPYT FGG GTKLEIK

The present application specifically includes and encompasses EphA2Binding Molecules (e.g., EphA2×CD3 bispecific Binding Molecules) thatare capable of binding to EphA2, and particularly such Binding Moleculesthat comprise the VL and/or VH Domain, and/or 1, 2 or all 3 of theCDR_(L)s of the VL Domain and/or 1, 2 or all 3 of the CDR_(H)s of the VHDomain of anti-EphA2 monoclonal antibodies EphA2 mAb 1, EphA2 mAb 2 andEphA2 mAb 3.

5. Exemplary Anti-gpA33 Antibodies

The 43 kD transmembrane glycoprotein A33 (gpA33) is expressed in >95% ofall colorectal carcinomas (Heath, J. K. et al. (1997) “The Human A33Antigen Is A Transmembrane Glycoprotein And A Novel Member Of TheImmunoglobulin Superfamily,” Proc. Natl. Acad. Sci. (U.S.A.)94(2):469-474; Ritter, G. et al. (1997) “Characterization OfPosttranslational Modifications Of Human A33 Antigen, A NovelPalmitoylated Surface Glycoprotein Of Human GastrointestinalEpithelium,” Biochem. Biophys. Res. Commun. 236(3):682-686; Wong, N. A.et al. (2006) “EpCAM and gpA33 Are Markers Of Barrett's Metaplasia,” J.Clin. Pathol. 59(3):260-263). An exemplary antibody that binds to humangpA33 is “gpA33 mAb 1.”

The amino acid sequence of the VH Domain of gpA33 mAb 1 (SEQ ID NO:146)is shown below (CDR_(H) residues are shown underlined):

QVQLVQSGAE VKKPGASVKV SCKASGYTFT  GSWMN WVRQA PGQGLEWIG R  IYPGDGETNY NGKFKD RVTI TADKSTSTAY MELSSLRSED TAVYYCAR IY   GNNVYFDVWG QGTTVTVSS

The amino acid sequence of the VL Domain of gpA33 mAb 1 (SEQ ID NO:147)is shown below (CDR_(L) residues are shown underlined):

DIQLTQSPSF LSASVGDRVT ITC SARSSIS FMY WYQQKPG KAPKLLIY DT   SNLASGVPSR FSGSGSGTEF TLTISSLEAE DAATYYC QQW SSYPLT FGQG TKLEIK

The present application specifically includes and encompasses gpA33Binding Molecules (e.g., gpA33×CD3 bispecific Binding Molecules) thatare capable of binding to gpA33, and particularly such Binding Moleculesthat comprise the VL and/or VH Domain, and/or 1, 2 or all 3 of theCDR_(L)s of the VL Domain and/or 1, 2 or all 3 of the CDR_(H)s of the VHDomain of anti-gpA33 monoclonal antibodies gpA33 mAb 1, or of any of theanti-gpA33 monoclonal antibodies provided in WO 2015/026894. The presentinvention additionally includes and encompasses the exemplary gpA33×CD3bispecific Binding Molecules provided in WO 2015/026894.

6. Exemplary Anti-HER2/neu Antibodies

HER2/neu is a 185 kDa receptor protein that was originally identified asthe product of the transforming gene from neuroblastomas of chemicallytreated rats. HER2/neu has been extensively investigated because of itsrole in several human carcinomas (including breast and gastric cancers)and in mammalian development (Hynes et al. (1994) Biochim. Biophys. Acta1198:165-184; Dougall et al. (1994) Oncogene 9:2109-2123; Lee et al.(1995) Nature 378:394-398). Exemplary antibodies that bind humanHER2/neu include “Margetuximab,” “Trastuzumab” and “Pertuzumab.”Margetuximab (also known as MGAH22; CAS Reg No. 1350624-75-7) is anFc-optimized monoclonal antibody that binds to HER2/neu and mediatesenhanced ADCC activity. Trastuzumab (also known as rhuMAB4D5, andmarketed as HERCEPTIN®; CAS Reg No 180288-69-1; see, U.S. Pat. No.5,821,337) is the humanized version of antibody 4D5, having IgG1/kappaconstant regions. Pertuzumab (also known as rhuMAB2C4, and marketed asPERJETA™; CAS Reg No 380610-27-5; see for example, WO2001/000245) is ahumanized version of antibody 2C4 having IgG1/kappa constant regions.

The present application specifically includes and encompasses Her2/Neubinding molecule (e.g., Her2/Neu×CD3 bispecific Binding Molecules) thatare capable of binding to Her2/Neu, and particularly such BindingMolecules that comprise the VL and/or VH Domain, and/or 1, 2 or all 3 ofthe CDR_(L)s of the VL Domain and/or 1, 2 or all 3 of the CDR_(H)s ofthe VH Domain of the anti-Her2/Neu monoclonal antibodies Margetuximab,Trastuzumab or Pertuzumab.

The amino acid sequence of the VH Domain of Margetuximab is (SEQ IDNO:148) (CDR_(H) residues are shown underlined):

QVQLQQSGPE LVKPGASLKL SCTASGFNIK  DTYIH WVKQR PEQGLEWIG R  IYPTNGYTRY DPKFQD KATI TADTSSNTAY LQVSRLTSED TAVYYCSR WG   GDGFYANDYW GQGASVTVSS

The amino acid sequence of the VL Domain of Margetuximab is (SEQ IDNO:149) (CDR_(L) residues are shown underlined):

DIVMTQSHKF MSTSVGDRVS ITC KASQDVN TAVA WYQQKP GHSPKLLIY S   ASFRYTGVPD RFTGSRSGTD FTFTISSVQA EDLAVYYC QQ HYTTPPT FGG GTKVEIK

The amino acid sequences of the complete Heavy and Light Chains ofMargetuximab are known in the art (see., e.g., WHO Drug Information,2014, Recommended INN: List 71, 28(1):93-94).

The amino acid sequence of the VH Domain of Trastuzumab is (SEQ IDNO:150) (CDR_(H) residues are shown underlined):

EVQLVESGGG LVQPGGSLRL SCAASGFNIK  DTYIH WVRQA PGKGLEWVA R  IYPTNGYTRY ADSVKG RFTI SADTSKNTAY LQMNSLRAED TAVYYCSR WG   GDGFYANDYW GQGTLVTVSS

The amino acid sequence of the VL Domain of Trastuzumab is (SEQ IDNO:151) (CDR_(L) residues are shown underlined):

DIQMTQSPSS LSASVGDRVT ITC RASQDVN TAVA WYQQKP GKAPKLLIY S   ASFLYSGVPS RFSGSRSGTD FTLTISSLQP EDFATYYC QQ HYTTPPT FGQ GTKVEIK

The amino acid sequence of the VH Domain of Pertuzumab is (SEQ IDNO:152) (CDR_(H) residues are shown underlined):

EVQLVESGGG LVQPGGSLRL SCAASGFTFT  DYTMD WVRQA PGKGLEWVA D  VNPNSGGSIY NQRFKG RFTL SVDRSKNTLY LQMNSLRAED TAVYYCAR NL   GPSFYFDYWG QGTLVTVSS

The amino acid sequence of the VL Domain of Pertuzumab is (SEQ IDNO:153) (CDR_(L) residues are shown underlined):

DIQMTQSPSS LSASVGDRVT ITC KASQDVS IGVA WYQQKP GKAPKLLIY S   ASYRYTGVPS RFSGSGSGTD FTLTISSLQP EDFATYYC QQ YYIYPYT FGQ GTKVEIK

In addition to the above-identified preferred anti-HER2/neu BindingMolecules, the invention contemplates Her2/Neu Binding Molecules thatcomprise the VL and/or VH Domain, and/or 1, 2 or all 3 of the CDR_(L)sof the VL Domain and/or 1, 2 or all 3 of the CDR_(H)s of the VH Domainof any of the following anti-Her-2 Binding Molecules: 1.44.1; 1.140;1.43; 1.14.1; 1.100.1; 1.96; 1.18.1; 1.20; 1.39; 1.24; and 1.71.3 (U.S.Pat. Nos. 8,350,011; 8,858,942; and PCT Patent Publication WO2008/019290); F5 and C1 (U.S. Pat. Nos. 7,892,554; 8,173,424; 8,974,792;and PCT Patent Publication WO 99/55367); and also the anti-Her-2 BindingMolecules of US Patent Publication US2013017114 and PCT PatentPublication Nos. WO2011/147986 and WO 2012/143524). The presentinvention additionally includes and encompasses the exemplaryHer2/Neu×CD3 bispecific Binding Molecules provided in WO 2012/143524.

7. Exemplary Anti-VEGF Antibodies

VEGF-A is a chemical signal that stimulates angiogenesis in a variety ofdiseases, especially in certain metastatic cancers such as metastaticcolon cancer, and in certain lung cancers, renal cancers, ovariancancers, and glioblastoma multiforme of the brain. An exemplary antibodythat binds to human VEGF-A is “Bevacizumab” (Avastin®). Bevacizumab is arecombinant humanized IgG1 monoclonal antibody (Midgley, R. et al.(2005) “Bevacizumab—Current Status And Future Directions,” Ann. Oncol.16(7):999-1004; Hall, R. D. et al. (2015) “Angiogenesis Inhibition As ATherapeutic Strategy In Non-Small Cell Lung Cancer (NSCLC),” Transl.Lung Cancer Res. 4(5):515-523; Narita, Y. (2015) “Bevacizumab ForGlioblastoma,” Ther. Clin. Risk Manag. 11:1759-1765).

The amino acid sequence of the VH Domain of Bevacizumab (SEQ ID NO:154)is shown below (CDR_(H) residues are shown underlined):

EVQLVESGGG LVQPGGSLRL SCAASGYTFT  NYGMN WVRQA PGKGLEWVG W  INTYTGEPTY AADFKR RFTF SLDTSKSTAY LQMNSLRAED TAVYYCA KYP   HYYGSSHWYF DVWGQGTLVT VSS

The amino acid sequence of the VL Domain of Bevacizumab (SEQ ID NO:155)is shown below (CDR_(L) residues are shown underlined):

DIQMTQSPSS LSASVGDRVT ITC SASQDIS NYLN WYQQKP GKAPKVLIY F   TSSLHSGVPS RFSGSGSGTD FTLTISSLQP EDFATYYC QQ YSTVPWT FGQ GTKVEIKR

The present application specifically includes and encompasses VEGFBinding Molecules (e.g., VEGF×CD3 bispecific Binding Molecules) that arecapable of binding to VEGF, and particularly such Binding Molecules thatcomprise the VL and/or VH Domain, and/or 1, 2 or all 3 of the CDR_(L)sof the VL Domain and/or 1, 2 or all 3 of the CDR_(H)s of the VH Domainof the anti-VEGF monoclonal antibody Bevacizumab.

8. Exemplary Anti-5T4 Antibodies

The oncofetal protein, 5T4, is a tumor-associated protein displayed onthe cell membrane of many carcinomas, including kidney, colon, prostate,lung, carcinoma and in acute lymphoblastic leukemia (see, Boghaert, E.R. et al. (2008) “The Oncofetal Protein, 5T4, Is A Suitable Target ForAntibody-Guided Anti-Cancer Chemotherapy With Calicheamicin,” Int. J.Oncol. 32(1):221-234; Eisen, T. et al. (2014) “Naptumomab Estafenatox:Targeted Immunotherapy with a Novel Immunotoxin,” Curr. Oncol. Rep.16:370, pp. 1-6). Exemplary antibodies that bind to human 5T4 include“5T4 mAb 1” and “5T4 mAb 2.”

The amino acid sequence of the VH Domain of 5T4 mAb 1 (SEQ ID NO:156) isshown below (CDR residues are shown underlined):

QVQLVQSGAE VKKPGASVKV SCKASGYTFT  SFWMH WVRQA PGQGLEWMG R   IDPNRGGTEY  NEKAKS RVTM TADKSTSTAY MELSSLRSED TAVYYCAG GN   PYYPMDY WGQ GTTVTVSS

The amino acid sequence of the VL Domain of an exemplary 5T4 mAb 1 (SEQID NO:157) is shown below (CDR residues are shown underlined):

DIQMTQSPSS LSASVGDRVT ITC RASQGIS   NYLA WFQQKP GKAPKSLIY R   ANRLQSGVPS RFSGSGSGTD FTLTISSLQP EDVATYYC LQ   YDDFPWT FGQ GTKLEIK

The amino acid sequence of the VH Domain of 5T4 mAb 2 (SEQ ID NO:158) isshown below (CDR residues are shown underlined):

QVQLQQPGAE LVKPGASVKM SCKASGYTFT  SYWIT WVKQR PGQGLEWIG D   IYPGSGRANY  NEKFKS KATL TVDTSSSTAY MQLSSLTSED SAVYNCAR YG   PLFTTVVDPN   SYAMDY WGQGTSVTVSS 

The amino acid sequence of the VL Domain of 5T4 mAb 2 (SEQ ID NO:159) isshown below (CDR residues are shown underlined):

DVLMTQTPLS LPVSLGDQAS ISC RSSQSIV   YSNGNTYLE W YLQKPGQSPK LLIY KVSNRF  S GVPDRFSGS GSGTDFTLKI SRVEAEDLGV YYC FQGSHVP   FT FGSGTKLE IK

The present application specifically includes and encompasses 5T4Binding Molecules (e.g., 5T4×CD3 bispecific Binding Molecules) that arecapable of binding to 5T4 that comprise the VL and/or VH Domain, and/or1, 2 or all 3 of the CDR_(L)s of the VL Domain and/or 1, 2 or all 3 ofthe CDR_(H)s of the VH Domain of the anti-5T4 monoclonal antibodies 5T4mAb 1 or 5T4 mAb 2, or of any of the anti-5T4 antibodies provided in WO2013/041687 or WO 2015/184203. The present invention additional includesand encompasses the exemplary 5T4×CD3 bispecific Binding Moleculesprovided in WO 2015/184203.

The present application additionally specifically includes andencompasses 5T4×CD3×CD8 trispecific Binding Molecules that are capableof binding to 5T4, to CD3 and to CD8, and particularly such trispecificBinding Molecules that comprise the VL and/or VH Domain, and/or 1, 2 orall 3 of the CDR_(L)s of the VL Domain and/or 1, 2 or all 3 of theCDR_(H)s of the VH Domain of the anti-5T4 monoclonal antibodies 5T4 mAb1 or 5T4 mAb 2 or of any of the anti-5T4 monoclonal antibodies providedin WO 2015/184203, and/or the VL and/or VH Domain, and/or 1, 2 or all 3of the CDR_(L)s of the VL Domain and/or 1, 2 or all 3 of the CDR_(H)s ofthe VH Domain of any of the anti-CD8 monoclonal antibodies providedherein.

9. Exemplary Anti-IL13Rα2 Antibodies

Interleukin-13 Receptor a2 (IL-13Ra2) is overexpressed in a variety ofcancers, including glioblastoma, colorectal cancer, cervical cancer,pancreatic cencer, multiple melanoma, osteosarcoma, leukemia, lymphoma,prostate cancer and lung cancer (PCT Pubmication No. WO 2008/146911;Brown, C. E. et al. (2013) “Glioma IL13Rα2 Is Associated WithMesenchymal Signature Gene Expression And Poor Patient Prognosis,” PLoSOne. 18; 8(10):e77769; Barderas, R. et al. (2012) “High Expression OfIL-13 Receptor A2 In Colorectal Cancer Is Associated With Invasion,Liver Metastasis, And Poor Prognosis,” Cancer Res. 72(11):2780-2790;Kasaian, M. T. et al. (2011) “IL-13 Antibodies Influence IL-13 ClearanceIn Humans By Modulating Scavenger Activity Of IL-13Rα2,” J. Immunol.187(1):561-569; Bozinov, O. et al. (2010) “Decreasing Expression Of TheInterleukin-13 Receptor IL-13Ralpha2 In Treated Recurrent MalignantGliomas,” Neurol. Med. Chir. (Tokyo) 50(8):617-621; Fujisawa, T. et al.(2009) “A Novel Role Of Interleukin-13 Receptor Alpha2 In PancreaticCancer Invasion And Metastasis,” Cancer Res. 69(22):8678-8685).Antibodies that immunospecifically bind to IL13Rα2 are commerciallyavailable and have been described in the art (Abnova Corporation,Biorbyt, LifeSpan BioSciences, United States Biologicals; see also PCTPublication No. WO 2008/146911). Exemplary antibodies that bind to humanIL-13Ra2 include “hu08” (see, e.g., PCT Publication No. WO 2014/072888).

The amino acid sequence of the VH Domain of hu08 (SEQ ID NO:160) isshown below (CDR residues are shown underlined):

EVQLVESGGG LVQPGGSLRL SCAASGFTFS  RNGMS WVRQA PGKGLEWVA T   VSSGGSYIYY  ADSVKG RFTI SRDNAKNSLY LQMNSLRAED TAVYYCAR QG   TTALATRFFD V WGQGTLVTVSS

The amino acid sequence of the VL Domain of hu08 (SEQ ID NO:161) isshown below (CDR residues are shown underlined):

DIQMTQSPSS LSASVGDRVT ITC KASQDVG   TAVA WYQQKP GKAPKLLIY S   ASYRSTGVPS RFSGSGSGTD FTLTISSLQP EDFATYYC QH   HYSAPWT FGG GTKVEIK

The present application specifically includes and encompasses IL13Rα2Binding Molecules (e.g., IL13Rα2×CD3 bispecific Binding Molecules) thatare capable of binding to IL13Rα2, and particularly such BindingMolecules that comprise the VL and/or VH Domain, and/or 1, 2 or all 3 ofthe CDR_(L)s of the VL Domain and/or 1, 2 or all 3 of the CDR_(H)s ofthe VH Domain of the anti-IL13Rα2 monoclonal antibody hu08.

10. Exemplary Anti-CD123 Antibodies

CD123 (interleukin 3 receptor alpha, IL-3Ra) is a 40 kDa molecule and ispart of the interleukin 3 receptor complex (Stomski, F. C. et al. (1996)“Human Interleukin-3 (IL-3) Induces Disulfide-Linked IL-3 ReceptorAlpha-And Beta-Chain Heterodimerization, Which Is Required For ReceptorActivation But Not High-Affinity Binding,” Mol. Cell. Biol.16(6):3035-3046). Interleukin 3 (IL-3) drives early differentiation ofmultipotent stem cells into cells of the erythroid, myeloid and lymphoidprogenitors. CD123 has been reported to be overexpressed on malignantcells in a wide range of hematologic malignancies including acutemyeloid leukemia (AML), chronic myelogenous leukemia (CML), acute Blymphoblastic leukemia (B-ALL), hairy cell leukemia (HCL), blasticplasmacytoid dendritic cell neoplasm (BPDCN), chronic myelogenousleukemia (CML), acute B lymphoblastic leukemia (B-ALL), hairy cellleukemia (HCL), blastic plasmacytoid dendritic cell neoplasm (BPDCN),and myelodysplastic syndrome (MDS) (Munoz, L. et al. (2001)“Interleukin-3 Receptor Alpha Chain (CD123) Is Widely Expressed InHematologic Malignancies,” Haematologica 86(12):1261-1269).Overexpression of CD123 is associated with poorer prognosis in AML(Tettamanti, M. S. et al. (2013) “Targeting Of Acute Myeloid LeukaemiaBy Cytokine-Induced Killer Cells Redirected With A Novel CD123-SpecificChimeric Antigen Receptor,” Br. J. Haematol. 161:389-401).

An exemplary antibody that binds to human CD123, and that may beemployed in the present invention, is “CD123 mAb 1” (see, e.g., PCTPatent Publication WO 2015/026892).

The amino acid sequence of the VH Domain of CD123 mAb 1 (SEQ ID NO:162)is shown below CDR_(H) residues are shown underlined):

EVQLVQSGAE LKKPGASVKV SCKASGYTFT  DYYMK WVRQA PGQGLEWIG D   IIPSNGATFY  NQKFKG RVTI TVDKSTSTAY MELSSLRSED TAVYYCAR SH   LLRASWFAY W GQGTLVTVSS

The amino acid sequence of the VL Domain of CD123 mAb 1 (SEQ ID NO:163)is shown below (CDR_(L) residues are shown underlined):

DFVMTQSPDS LAVSLGERVT MSC KSSQSLL NSGNQKNYLT WYQQKPGQPP KLLIY WASTR ESGVPDRFSG SGSGTDFTLT ISSLQAEDVA VYYC QNDYSY   PYT FGQGTKL EIK

The present application specifically includes and encompasses CD123Binding Molecules (e.g., CD123×CD3 bispecific Binding Molecules) thatare capable of binding to CD123, and particularly such Binding Moleculesthat comprise the VL and/or VH Domain, and/or 1, 2 or all 3 of theCDR_(L)s of the VL Domain and/or 1, 2 or all 3 of the CDR_(H)s of the VHDomain of the anti-CD123 monoclonal antibody CD123 mAb 1, and also anyof the anti-CD123 antibodies disclosed in US 2017/081424 and WO2016/036937. The present invention additionally includes and encompassesexemplary CD123×CD3 bispecific Binding Molecules, including:flotetuzumab (aka MGD007; CAS Registry No. 1664355-28-5), JNJ-63709178(Johnson & Johnson, also see, WO 2016/036937) and XmAb14045 (Xencor,also see, US 2017/081424).

11. Exemplary Anti-CD19 Antibodies

CD19 (B lymphocyte surface antigen B4, Genbank accession number M28170)is a component of the B-cell-receptor (BCR) complex, and is a positiveregulator of B-Cell signaling that modulates the threshold for B-Cellactivation and humoral immunity. CD19 is one of the most ubiquitouslyexpressed antigens in the B-Cell lineage and is expressed on >95% ofB-Cell malignancies, including acute lymphoblastic leukemia (ALL),chronic lymphocytic leukemia (CLL), and non-Hodgkin's Lymphoma (NHL).Notably, CD19 expression is maintained on B-Cell lymphomas that becomeresistant to anti-CD20 therapy (Davis et al. (1999) “Therapy of B-CellLymphoma With Anti-CD20 Antibodies Can Result In The Loss Of CD20Antigen Expression.” Clin Cancer Res, 5:611-615, 1999). CD19 has alsobeen suggested as a target to treat autoimmune diseases (Tedder (2009)“CD19: A Promising B-Cell Target For Rheumatoid Arthritis,” Nat. Rev.Rheumatol. 5:572-577).

An exemplary humanized antibody that binds to human CD19, and that maybe employed in the present invention, is the anti-CD19 antibody diclosedin WO 2016/048938 (referred to herein as “CD19 mAb 1”).

The amino acid sequence of the VH Domain of CD19 mAb 1 (SEQ ID NO:164)is shown below (CDR_(H) residues are shown underlined):

QVTLRESGPA LVKPTQTLTL TCTFSGFSLS  TSGMGVG WIR QPPGKALEWL A HIWWDDDKR  YNPALKS RLT ISKDTSKNQV FLTMTNMDPV DTATYYCAR M   ELWSYYFDY W GQGTTVTVSS

The amino acid sequence of the VL Domain of CD19 mAb 1 (SEQ ID NO:165)is shown below CDR_(L) residues are shown underlined:

ENVLTQSPAT LSVTPGEKAT ITC RASQSVS YMH WYQQKPG QAPRLLIY DA   SNRASGVPSR FSGSGSGTDH TLTISSLEAE DAATYYC FQG SVYPF TFGQG TKLEIK

The amino acid sequence of an alternative VL Domain of CD19 mAb 1 (SEQID NO:195) is shown below (CDR_(L) residues are shown underlined):

ENVLTQSPAT LSVTPGEKVT ITC SASSSVS YMH WYQQKPG QAPRLLIY DT   SKLASGVPSR FSGSGSGTDH FLTISSLEAE DAATYYC FQG SVYPFT FGQG TKLEIK

The present application specifically includes and encompasses CD19Binding Molecules (e.g., CD19×CD3 bispecific Binding Molecules) that arecapable of binding to CD19, and particularly such Binding Molecules thatcomprise the VL and/or VH Domain, and/or 1, 2 or all 3 of the CDR_(L)sof the VL Domain and/or 1, 2 or all 3 of the CDR_(H)s of the VH Domainof the anti-CD19 monoclonal antibody CD19 mAb 1, or any of the anti-CD19antibodies disclosed in U.S. Pat. No. 7,112,324. The present inventionspecifically includes and encompasses exemplary CD19×CD3 bispecificBinding Molecules that may be employed in the present invention,including: blinatumomab (BLINCYTO®; amino acid sequence found in WHODrug Information, 2009, Recommended INN: List 62, 23(3):240-241) andduvortuxizumab (aka MGD011; amino acid sequence found in WHO DrugInformation, 2016, Proposed INN: List 116, 30(4):627-629).

B. Exemplary Pathogen-Associated Antigens

As used herein, the term “Pathogen Antigen” denotes an antigen that ischaracteristically expressed on the surface of a pathogen-infected cell,and that may thus be treated with an Antibody-Based Molecule or anImmunomodulatory Molecule. Examples of Pathogen Antigens include, butare not limited to antigens expressed on the surface of a cell infectedwith: a Herpes Simplex Virus (e.g., infected cell protein (ICP)47, gD,etc.), a varicella-zoster virus, a Kaposi's sarcoma-associatedherpesvirus, an Epstein-Barr Virus (e.g., LMP-1, LMP-2A, LMP-2B, etc.),a Cytomegalovirus (e.g., UL11, etc.), Human Immunodeficiency Virus(e.g., env proteins gp160, gp120, gp41, etc.), a Human Papillomavirus(e.g., E6, E7, etc.), a human T-cell leukemia virus (e.g., env proteinsgp64, gp46, gp21, etc.), Hepatitis A Virus, Hepatitis B Virus, HepatitisC Virus, Vesicular Stomatitis Virus (VSV), Bacilli, Citrobacter,Cholera, Diphtheria, Enterobacter, Gonococci, Helicobacter pylori,Klebsiella, Legionella, Meningococci, mycobacteria, Pseudomonas,Pneumonococci, rickettsia bacteria, Salmonella, Serratia, Staphylococci,Streptococci, Tetanus, Aspergillus (fumigatus, niger, etc.), Blastomycesdermatitidis, Candida (albicans, krusei, glabrata, tropicalis, etc.),Cryptococcus neoformans, Genus Mucorales (mucor, absidia, rhizopus),Sporothrix schenkii, Paracoccidioides brasiliensis, Coccidioidesimmitis, Histoplasma capsulatum, Leptospirosis, Borrelia burgdorferi,helminth parasite (hookworm, tapeworms, flukes, flatworms (e.g.Schistosomia), Giardia lambia, trichinella, Dientamoeba Fragilis,Trypanosoma brucei, Trypanosoma cruzi, and Leishmania donovani). Suchantibodies are available commercially from a wide number of sources, orcan be obtained by immunizing mice or other animals (including for theproduction of monoclonal antibodies) with such antigens.

Exemplary antibodies, whose VH and VL Domains may be used to constructmolecules capable of binding a Pathogen Antigen arrayed on the surfaceof a pathogen-infected cell are antibodies are provided below,additional antibodies are known in the art.

1. Exemplary Anti-HIV Env Antibody

The env protein of HIV is an exemplary Pathogen-Associated Antigen, andantibodies that bind the env protein of HIV are exemplary of antibodiescapable of binding a Pathogen-Associated Antigen.

The initial step in HIV-1 infection occurs with the binding of cellsurface CD4 to trimeric HIV-1 envelope glycoproteins (env), aheterodimer of a transmembrane glycoprotein (gp41) and a surfaceglycoprotein (gp120). The gp120 and gp41 glycoproteins are initiallysynthesized as a single gp160 polypeptide that is subsequently cleavedto generate the non-covalently associated gp120/gp4l complex. Theectodomain of env is a heterodimer with mass of approximately 140 kDa,composed of the entire gp120 component, and approximately 20 kDa of gp41(Harris, A. et al. (2011) “Trimeric HIV-1 Glycoprotein Gp140 ImmunogensAnd Native HIV-1 Envelope Glycoproteins Display The Same Closed And OpenQuaternary Molecular Architectures,” Proc. Natl. Acad. Sci. (U.S.A.)108(28):11440-11445). Antibodies that that immunospecifically bind toenv proteins are commercially available and have been described in theart (see, e.g., GenBank Accession No. AFQ31503; Buchacher, A. et al.(1994) “Generation Of Human Monoclonal Antibodies Against HIV-1Proteins; Electrofusion And Epstein-Barr Virus Transformation ForPeripheral Blood Lymphocyte Immortalization,” AIDS Res. Hum.Retroviruses 10(4):359-369; Shen, R. (2010) “GP41-Specific AntibodyBlocks Cell-Free HIV Type 1 Transcytosis Through Human Rectal Mucosa AndModel Colonic Epithelium,” J. Immunol. 184(7):3648-3655; WO 2012/162068;and WO 2016/054101). Exemplary antibodies that bind to HIV env include“7B2” (GenBank Accession No. AFQ31503) and “A32” (PCT Publication No. WO2014/159940). Multiple VH Domains of Antibody A32 have been reported inthe art that possess minor changes in framework regions 1 and/or 4reported (see, e.g., Protein Data Base Accession number PDB: 4YBL_H, US2015/0239961 and WO 2006/044410). Any of these variant Antibody A32 VHDomains may be employed in accordance with the present invention.

The amino acid sequence of the VH Domain of 7B2 (SEQ ID NO:166) is shownbelow (CDR residues are shown underlined):

QVQLVQSGGG VFKPGGSLRL SCEASGFTFT  EYYMT WVRQA PGKGLEWLAY  ISKNGEYSKY  SPSSNG RFTI SRDNAKNSVF LQLDRLSADD TAVYYCAR AD   GLTYFSELLQ   YIFDL WGQGARVTVSS

The amino acid sequence of the VL Domain of 7B2 (SEQ ID NO:167) is shownbelow (CDR residues are shown underlined):

DIVMTQSPDS LAVSPGERAT IHCK SSQTLL   YSSNNRHSIA WYQQRPGQPP KLLLY WASMR  LS GVPDRFSG SGSGTDFTLT INNLQAEDVA IYYC HQYSSH   PPT FGHGTRV EIK

The amino acid sequence of an exemplary VH Domain of A32 (SEQ ID NO:168)is shown below (CDR residues are shown underlined):

QVQLQESGPG LVKPSQTLSL SCTVSGGSSS  SGAHYWS WIR QYPGKGLEWI G YIHYSGNTY  YNPSLKS RIT ISQHTSENQF SLKLNSVTVA DTAVYYCAR G   TRLRTLRNAF DI WGQGTXVTVSS

-   -   wherein: X is L or M

The amino acid sequence of such an exemplary VH Domain of A32 (SEQ IDNO:209), wherein X is L, is shown below (CDR residues are shownunderlined):

QVQLQESGPG LVKPSQTLSL SCTVSGGSSS  SGAHYWS WIR QYPGKGLEWI G YIHYSGNTY  YNPSLKS RIT ISQHTSENQF SLKLNSVTVA DTAVYYCAR G   TRLRTLRNAF DI WGQGTLVTVSS

The amino acid sequence of the VL Domain of A32 (SEQ ID NO:169) is shownbelow (CDR residues are shown underlined):

QSALTQPPSA SGSPGQSVTI SC TGTSSDVG GYNYVS WYQH HPGKAPKLII S EVNNRPSGV PDRFSGSKSG NTASLTVSGL QAEDEAEYYC  SSYTDIHNFV  FGGGTKLTVL

The present application specifically includes and encompasses HIVBinding Molecules (e.g., HIV×CD3 bispecific Binding Molecules) that arecapable of binding to HIV, and particularly such Binding Molecules thatcomprise the VL and/or VH Domain, and/or 1, 2 or all 3 of the CDR_(L)sof the VL Domain and/or 1, 2 or all 3 of the CDR_(H)s of the VH Domainof the anti-HIV monoclonal antibodies 7B2, A32, and also any of theanti-HIV antibodies disclosed in WO 2016/054101, WO 2017/011413, WO2017/011414. The present invention specifically includes and encompassesthe exemplary HIV×CD3 bispecific Binding Molecules provided in WO2014/159940, WO 2015/184203, WO 2017/011413, and WO 2017/011414.

The present application additionally specifically includes andencompasses HIV×CD3×CD8 trispecific Binding Molecules that are capableof binding to HIV, to CD3 and to CD8, and particularly such trispecificBinding Molecules that comprise the VL and/or VH Domain, and/or 1, 2 orall 3 of the CDR_(L)s of the VL Domain and/or 1, 2 or all 3 of theCDR_(H)s of the VH Domain of the anti-HIV monoclonal antibodies 7B2 orA32 or of any of the anti-HIV monoclonal antibodies provided in WO2015/184203, WO 2016/054101, WO 2017/011413, WO 2017/011414, and/or theVL and/or VH Domain, and/or 1, 2 or all 3 of the CDR_(L)s of the VLDomain and/or 1, 2 or all 3 of the CDR_(H)s of the VH Domain of any ofthe anti-CD8 monoclonal antibodies provided in WO 2015/184203.

2. Exemplary Anti-RSV Glycoprotein F Antibody

A further illustrative Pathogen-Associated Antigen is RSV glycoproteinF. An exemplary anti-RSV glycoprotein F antibody is palivizumab (see,e.g., Protein Data Bank (PDB) ID No. 2HWZ). Alternative anti-RSVglycoprotein F antibodies include motavizumab (see, e.g., PDB ID No.3IXT) and a variant of palivizumab that has been engineered to remove acysteine residue from palivizumab's CDR_(L)1. The amino acid sequence ofthe VH Domain of the variant of palivizumab (SEQ ID NO:170) is shownbelow (CDR residues are shown underlined):

QVTLRESGPA LVKPTQTLTL TCTFSGFSLS  TSGMSVG WIR QPPGKALEWL A DIWWDDKKD  YNPSLKS RLT ISKDTSKNQV VLKVTNMDPA DTATYYCAR S   MITNWYFDV W GAGTTVTVSS

The amino acid sequence of the VL Domain of the variant of palivizumab(SEQ ID NO:171) is shown below (CDR residues are shown underlined):

DIQMIQSPST LSASVGDRVT ITC RASQSVG YMH WYQQKPG KAPKLLIY DT   SKLASGVPSR FSGSGSGTEF TLTISSLQPD DFATYYC FQG SGYPFT FGGG TKLEIK

VII. Exemplary Binding Molecules of the Present Invention

As discussed below, the present invention is illustrated using severalDA×CD3 Binding Molecules having different structures including moleculescapable of mediating the redirected killing of a tumor cell (e.g., a“DART-A”-type diabody or a “DART-B”-type diabody or a TRIVALENT-typemolecule, as described below).

A. DART-A-Type Diabodies

DART-A-type diabodies are bispecific diabodies capable of binding CD3and a Disease Antigen (e.g., a Cancer Antigen) that do not comprise anFc Domain. Provided herein are illustrative DART-A-type diabodiescomposed of two polypeptide chains having one binding site for CD3 andone binding site for the Cancer Antigen CD123 (see, e.g., FIG. 1 ).

An illustrative DART-A-type diabody (designated “DART-A-WT”) has a firstpolypeptide chain having the amino acid sequence of SEQ ID NO:172:

DFVMTQSPDS LAVSLGERVT MSCKSSQSLLNSGNQKNYLT WYQQKPGQPP KLLIYWASTR ESGVPDRFSGSGSGTDFTLT ISSLQAEDVA VYYCQNDYSY PYTFGQGTKL EIK 

EVQLVESGG GLVQPGGSLR LSCAASGFTFSTYAMNWVRQ APGKGLEWVG RIRSKYNNYA TYYADSVK 

R FTISRDDSKN SLYLQMNSLK TEDTAVYYCV RHGNFGNSYV SWFAYWGQGT LVTVSS GGCG  GG KVAALKEK VAALKEKVAA LKEKVAALKE

Residues 1-113 of the first polypeptide chain of such illustrativeDART-A-type diabody correspond to the VL Domain of CD123 mAb 1 (SEQ IDNO:162). Residues 114-121 (double underlined) of the first polypeptidechain of such illustrative DART-A-type diabody correspond to Linker 1(GGGSGGGG; SEQ ID NO:16). Residues 122-246 of the first polypeptidechain of such illustrative DART-A-type diabody correspond to the VHDomain of CD3 mAb 1 (SEQ ID NO:55), wherein Kabat position 65 (doubleunderlined) is aspartate (D). Residues 247-252 (underlined) of the firstpolypeptide chain of such illustrative DART-A-type diabody correspond toa Linker 2 (GGCGGG; SEQ ID NO:17). Residues 253-280 of the firstpolypeptide chain of such illustrative DART-A-type diabody correspond tothe heterodimer-promoting “K-coil” (KVAALKE-KVAALKE-KVAALKE-KVAALKE; SEQID NO:30).

The second polypeptide chain of such illustrative DART-A-type diabodyDART-A-WT has the amino acid sequence of SEQ ID NO:173:

QAVVTQEPSL TVSPGGTVTL TCRSSTGAVT TSNYANWVQQKPGQAPRGLI GGTNKRAPWT PARFSGSLLG GKAALTITGAQAEDEADYYC ALWYSNLWVF GGGTKLTVLG 

EV QLVQSGAELK KPGASVKVSC KASGYTFTDY YMKWVRQAPGQGLEWIGDII PSNGATFYNQ KFKGRVTITV DKSTSTAYMELSSLRSEDTA VYYCARSHLL RASWFAYWGQ GTLVTVSS GG CGGGEVAALE KEVAALEKEV AALEKEVAAL EK

Residues 1-110 of the second polypeptide chain of such illustrativeDART-A-type diabody DART-A-WT correspond to the VL Domain of CD3 mAb 1(SEQ ID NO:56). Residues 111-118 (double underlined) of the secondpolypeptide chain of such illustrative DART-A-type diabody correspond toLinker 1 (GGGSGGGG; SEQ ID NO:16). Residues 119-238 of the secondpolypeptide chain of such illustrative DART-A-type diabody correspond tothe VH Domain of CD123 mAb 1 (SEQ ID NO:163). Residues 239-244(underlined) of the second polypeptide chain of such illustrativeDART-A-type diabody correspond to Linker 2 (GGCGGG; SEQ ID NO:17).Residues 245-272 of the second polypeptide chain of such illustrativeDART-A-type diabody correspond to the heterodimer-promoting “E-coil”(EVAALEK-EVAALEK-EVAALEK-EVAALEK; SEQ ID NO:29).

As will be recognized in view of the instant disclosure, additionalDART-A-type diabodies having a binding site for an alternative DiseaseAntigen and/or having the CD3 Binding Domains of a variant anti-CD3antibody (i.e., a vCD3-Binding Domain) may likewise be constructed (byemploying the VL and VH Domains of such antibodies in lieu of the VL andVH Domains of the illustrative DART-A-type diabody). Similarly, asprovided herein, alternative DART-A-type molecules may likewise beconstructed incorporating alternative Linkers and/or alternativeHeterodimer-Promoting Domains. For example, an illustrative panel ofCD123×CD3 DART-A-type diabodies were generated having the same structureas DART-A-WT diabody provided above, but comprising the VL and VHDomains of one of the CD3 mAb 1 variants (M1-M26) provided above.

Each illustrative CD123×CD3 DART-A-type diabody of the panel has a firstpolypeptide chain having the amino acid sequence of SEQ ID NO: SEQ IDNO:189:

DFVMTQSPDS LAVSLGERVT MSCKSSQSLL NSGNQKNYLTWYQQKPGQPP KLLIYWASTR ESGVPDRFSG SGSGTDFTLTISSLQAEDVA VYYCQNDYSY PYTFGQGTKL EIK 

EVQLVESGG GLVQPGGSLR LSCAASGFTF SX ₁ X ₂ X ₃MNWVRQAPGKGLEWVG RIRSKYNNYA TYYADSVKX ₄R FTISRDDSKN SLYLQMNSLK TEDTAVYYCV RHX₅NX ₆ X ₇NSX ₈V  X ₉ X ₁₀FAX ₁₁WGQGT LVTVSS GGCG   GGKVAALKEK VAALKEKVAA LKEKVAALKEwherein: X₁ is T, D, or E; X₂ is Y, D or T; X₃ is A or G; X₄ is D or G;X₅ is G, D, E, or K; X₆ is F or I; X₇ is G or I; X₈ is Y, A, G, or Q; X₉is S or T; X₁₀ is W, F, or Y; and X₁₁ is Y or E.

Residues 1-113 of the first polypeptide chain of the panel ofillustrative DART-A-type diabodies correspond to the VL Domain of CD123mAb 1 (SEQ ID NO:162). Residues 114-121 (double underlined) of the firstpolypeptide chain of the panel of illustrative DART-A-type diabodiescorrespond to Linker 1 (GGGSGGGG; SEQ ID NO:16; double underlined).Residues 122-246 of the first polypeptide chain of the panel ofillustrative DART-A-type diabodies correspond to the VH Domain of CD3mAb 1 M1-CD3 mAb 1 M22 (SEQ ID NOs: 64, 66, 68, 70, 72, 74, 76, 78, 80,82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104 or 106). Residues247-252 (single underlined) of the panel of illustrative DART-A-typediabodies correspond to a Linker 2 (GGCGGG; SEQ ID NO:17). Residues253-280 of the first polypeptide chain of the panel of illustrativeDART-A-type diabodies correspond to the heterodimer-promoting “K-coil”(KVAALKE-KVAALKE-KVAALKE-KVAALKE; SEQ ID NO:30).

The second polypeptide chain of such illustrative DART-A-type diabodyhas the amino acid sequence of SEQ ID NO:190:

QAVVTQEPSL TVSPGCTVTL TCRSSTGAVT TSNYANWVQQ KPGQAPRGLI GX ₁TNX₂RAPWT PARFSGSLLG GKAALTITGA QAEDEADYYC AX ₃WYSNLWVF GGGTKLTVLG 

EV QLVQSGAELK KPGASVKVSC KASGYTFTDY YMKWVRQAPGQGLEWIGDII PSNGATFYNQ KFKGRVTITV DKSTSTAYMELSSLRSEDTA VYYCARSHLL RASWFAYWGQ GTLVTVSS GG CGGGEVAALE KEVAALEKEV AALEKEVAAL EKwherein: X₁ is G or D; X₂ is K or G; and X₃ is L, E or Q.

Residues 1-110 of the second polypeptide chain of the panel ofillustrative DART-A-type diabodies correspond to the VL Domain of CD3mAb 1 M23-CD3 mAb 1 M26 (SEQ ID NOs:108, 110, 112, and 114). Residues111-118 (double underlined) of the second polypeptide chain of the panelof illustrative DART-A-type diabodies correspond to Linker 1 (GGGSGGGG;SEQ ID NO:16; double underlined). Residues 119-238 of the secondpolypeptide chain of the panel of illustrative DART-A-type diabodiescorrespond to the VH Domain of CD123 mAb 1 (SEQ ID NO:163). Residues239-244 (underlined) of the second polypeptide chain of the panel ofillustrative DART-A-type diabodies correspond to Linker 2 (GGCGGG; SEQID NO:17; single underlined). Residues 245-272 of the second polypeptidechain of the panel of illustrative DART-A-type diabodies correspond tothe heterodimer-promoting “E-coil” (EVAALEK-EVAALEK-EVAALEK-EVAALEK; SEQID NO:29).

The amino acid sequences and designations of the panel of illustrativeDART-A-type diabodies comprising the VL and VH of the CD3 mAb 1 variantsare provided in Table 8 below.

TABLE 8 Illustrative DART-A-Type Diabodies First Polypeptide ChainSecond Polypeptide Chain Designation SEQ ID NO: SEQ ID NO. DART-A-M1189 - wherein: X₁ is T; X₂ is Y; X₃ is A; 173 X₄ is D; X₅ is G; X₆ is F;X₇ is G; X₈ is Y; X₉ is T; X₁₀ is W; and X₁₁ is Y DART-A-M2 189 -wherein: X₁ is T; X₂ is Y; X₃ is A; 173 X₄ is D; X₅ is K; X₆ is F; X₇ isG; X₈ is Y; X₉ is T; X₁₀ is W; and X₁₁ is Y DART-A-M3 189 - wherein: X₁is T; X₂ is Y, X₃ is A; 173 X₄ is D; X₅ is G; X₆ is F; X₇ is I; X₈ is Y;X₉ is S; X₁₀ is W; and X₁₁ is Y DART-A-M4 189 - wherein: X₁ is T; X₂ isY; X₃ is A; 173 X₄ is D; X₅ is G; X₆ is F; X₇ is G; X₈ is A; X₉ is S;X₁₀ is W; and X₁₁ is Y DART-A-M5 189 - wherein: X₁ is T; X₂ is Y; X₃ isA; 173 X₄ is D; X₅ is G; X₆ is F; X₇ is G; X₈ is G; X₉ is S; X₁₀ is W;and X₁₁ is Y DART-A-M6 189 - wherein: X₁ is T; X₂ is Y; X₃ is A; 173 X₄is D; X₅ is G; X₆ is F; X₇ is G; X₈ is Q; X₉ is S; X₁₀ is W; and X₁₁ isY DART-A-M7 189 - wherein: X₁ is T; X₂ is Y; X₃ is A; 173 X₄ is D; X₅ isD; X₆ is F; X₇ is G; X₈ is Y; X₉ is S; X₁₀ is W; and X₁₁ is Y DART-A-M8189 - wherein: X₁ is T; X₂ is Y, X₃ is A; 173 X₄ is D; X₅ is E; X₆ is F;X₇ is G; X₈ is Y; X₉ is S; X₁₀ is W; and X₁₁ is Y DART-A-M9 189 -wherein: X₁ is T; X₂ is Y; X₃ is A; 173 X₄ is D; X₅ is K; X₆ is F; X₇ isG; X₈ is Y; X₉ is S; X₁₀ is W; and X₁₁ is Y DART-A-M10 189 - wherein: X₁is T; X₂ is Y; X₃ is A; 173 X₄ is D; X₅ is G; X₆ is I; X₇ is G; X₈ is Y;X₉ is S; X₁₀ is W; and X₁₁ is Y DART-A-M11 189 - wherein: X₁ is T; X₂ isY; X₃ is A; 173 X₄ is D; X₅ is G; X₆ is F; X₇ is G; X₈ is Y; X₉ is S;X₁₀ is F; and X₁₁ is Y DART-A-M12 189 - wherein: X₁ is T; X₂ is Y; X₃ isA; 173 X₄ is D; X₅ is G; X₆ is F; X₇ is G; X₈ is Y; X₉ is S; X₁₀ is Y;and X₁₁ is Y DART-A-M13 189 - wherein: X₁ is T; X₂ is Y; X₃ is A; 173 X₄is D; X₅ is G; X₆ is F; X₇ is G; X₈ is Y; X₉ is S; X₁₀ is W; and X₁₁ isE DART-A-M14 189 - wherein: X₁ is D; X₂ is Y; X₃ is A; 173 X₄ is D; X₅is G; X₆ is F; X₇ is G; X₈ is Y; X₉ is S; X₁₀ is W; and X₁₁ is YDART-A-M15 189 - wherein: X₁ is E; X₂ is Y; X₃ is A; 173 X₄ is D; X₅ isG; X₆ is F; X₇ is G; X₈ is Y; X₉ is S; X₁₀ is W; and X₁₁ is Y DART-A-M16189 - wherein: X₁ is T; X₂ is D; X₃ is A; 173 X₄ is D; X₅ is G; X₆ is F;X₇ is G; X₈ is Y; X₉ is S; X₁₀ is W; and X₁₁ is Y DART-A-M17 189 -wherein: X₁ is T; X₂ is T; X₃ is A; 173 X₄ is D; X₅ is G; X₆ is F; X₇ isG; X₈ is Y; X₉ is S; X₁₀ is W; and X₁₁ is Y DART-A-M18 189 - wherein: X₁is T; X₂ is Y; X₃ is G; 173 X₄ is D; X₅ is G; X₆ is F; X₇ is G; X₈ is Y;X₉ is S; X₁₀ is W; and X₁₁ is Y DART-A-M19 189 - wherein: X₁ is T; X₂ isY; X₃ is A; 173 X₄ is D; X₅ is K; X₆ is I; X₇ is G; X₈ is Y; X₉ is S;X₁₀ is W; and X₁₁ is Y DART-A-M20 189 - wherein: X₁ is T; X₂ is Y; X₃ isA; 173 X₄ is D; X₅ is K; X₆ is F; X₇ is G; X₈ is G; X₉ is S; X₁₀ is W;and X₁₁ is Y DART-A-M21 189 - wherein: X₁ is T; X₂ is Y; X₃ is A; 173 X₄is D; X₅ is K; X₆ is F; X₇ is G; X₈ is Y; X₉ is S; X₁₀ is F; and X₁₁ isY DART-A-M22 189 - wherein: X₁ is T; X₂ is Y, X₃ is A; 173 X₄ is D; X₅is K; X₆ is F; X₇ is G; X₈ is Y; X₉ is S; X₁₀ is Y; and X₁₁ is YDART-A-M23 172 190 - wherein: X₁ is G; X₂ is K; and X₃ is E DART-A-M24172 190 - wherein: X₁ is G; X₂ is K; and X₃ is Q DART-A-M25 172 190 -wherein: X₁ is D; X₂ is K; and X₃ is L DART-A-M26 172 190 - wherein: X₁is G; X₂ is G; and X₃ is L

B. DART-B-Type Diabodies

DART-B-type diabodies are bispecific diabodies capable of binding CD3and a Disease Antigen (e.g., a Cancer or Infectious Disease Antigen)that comprise an Fc Domain. Provided herein are illustrative DART-B-typediabodies (Table 9) composed of three polypeptide chains and have onebinding site for CD3 and one binding site for the Cancer Antigen CD123,5T4, or CD19 (see, e.g., FIG. 4A).

TABLE 9 Disease Polypeptide Chain DART- Antigen- CD3 First Second B-TypeBinding Binding CD123/CD3 Third No. Domain Domain Binding Domains FcDomain Designation 1 CD 123 CD3 mAb 1 SEQ ID SEQ ID SEQ ID CD123-WT mAb1 NO: 174 NO: 175 NO: 176 2 CD3 mAb 1 SEQ ID CD123-M1 M1 NO: 177 3 CD3mAb 1 SEQ ID CD123-M2 M2 NO: 178 4 CD3 mAb 1 SEQ ID CD123-M18 M18 NO:179 5 CD3 mAb 1 SEQ ID CD123-M13 M13 NO: 198 6 CD3 mAb 1 SEQ IDCD123-M17 M17 NO: 199 7 CD3 mAb 1 SEQ ID CD123-M19 M19 NO: 200 8 5T4 CD3mAb 1 SEQ ID SEQ ID 5T4-WT mAb 1 NO: 180 NO: 181 9 CD3 mAb 1 SEQ ID5T4-M1 M1 NO: 182 10 CD3 mAb 1 SEQ ID 5T4-M2 M2 NO: 183 11 CD3 mAb 1 SEQID 5T4-M18 M18 NO: 184 12 HIV CD3 mAb 1 SEQ ID SEQ ID HIV-WT mAb A32 NO:185 NO: 186 13 CD3 mAb 1 SEQ ID HIV-M18 M18 NO: 196 14 CD19 CD3 mAb 1SEQ ID SEQ ID CD19-WT mAb 1 NO: 191 NO: 192 15 (alternative VL CD3 mAb 1SEQ ID CD19-M18 where indicated) M18 NO: 197 16 CD3 mAb 1 SEQ ID SEQ IDCD19.1-M18 M18 NO: 193 NO: 194 17 CD3 mAb 1 SEQ ID CD19.1-M13 M13 NO:201 18 CD3 mAb 1 SEQ ID CD19.1-M17 M17 NO: 202 19 CD3 mAb 1 SEQ IDCD19.1-M19 M19 NO: 203

1. First Illustrative DART-B-Type Diabody CD123-WT (CD123×CD3 mAb 1)

A first illustrative DART-B-type diabody (designated “CD123-WT”) has afirst polypeptide chain having the amino acid sequence of SEQ ID NO:174:

DFVMTQSPDS LAVSLGERVT MSCKSSQSLL NSGNQKNYLTWYQQKPGQPP KLLIYWASTR ESGVPDRFSG SGSGTDFTLTISSLQAEDVA VYYCQNDYSY PYTFGQGTKL EIK 

EVQLVESGG GLVQPGGSLR LSCAASGFTFSTYAMNWVRQ APGKGLEWVG RIRSKYNNYA TYYADSVK 

R FTISRDDSKN SLYLQMNSLK TEDTAVYYCV RHGNFGNSYV SWFAYWGQGT LVTVSS GGCG  GG EVAALEKE VAALEKEVAA LEKEVAALEK GGG DKTHTCP PCP APEAAGG PSVFLFPPKPKDTLMISRTP EVTCVVVDVS HEDPEVKFNW YVDGVEVHNAKTKPREEQYN STYRVVSVLT VLHQDWLNGK EYKCKVSNKALPAPIEKTIS KAKGQPREPQ VYTLPPSREE MTKNQVSLWCLVKGFYPSDI AVEWESNGQP ENNYKTIPPV LDSDGSFFLYSKLTVDKSRW QQGNVFSCSV MHEALHNHYT QKSLSLSPGK

Residues 1-113 of the first polypeptide chain of CD123-WT correspond tothe VL Domain of CD123 mAb 1 (SEQ ID NO:163). Residues 114-121 (doubleunderlined) of the first polypeptide chain of CD123-WT correspond toLinker 1 (GGGSGGGG; SEQ ID NO:16). Residues 122-246 of the firstpolypeptide chain of CD123-WT correspond to the VH Domain of CD3 mAb 1(SEQ ID NO:55), wherein Kabat position 65 (double underlined) isaspartate (D). Residues 247-252 (underlined) of the first polypeptidechain of CD123-WT correspond to a Linker 2 (GGCGGG; SEQ ID NO:17).Residues 253-280 of the first polypeptide chain of CD123-WT correspondto the heterodimer-promoting “E-coil” (EVAALEK-EVAALEK-EVAALEK-EVAALEK;SEQ ID NO:29). Residues 281-283 of the first polypeptide chain ofCD123-WT correspond to a GGG Linker. Residues 284-293 (underlined) ofthe first polypeptide chain of CD123-WT correspond to the LinkerDKTHTCPPCP (SEQ ID NO:40). Residues 294-510 of the first polypeptidechain of CD123-WT correspond to the IgG1 “knob-bearing” CH2-CH3 Domain(SEQ ID NO:48).

The second polypeptide chain of CD123-WT has the amino acid sequence ofSEQ ID NO:175:

QAVVTQEPSL TVSPGGTVTL TCRSSTGAVT TSNYANWVQQKPGQAPRGLI GGTNKRAPWT PARFSGSLLG GKAALTITGAQAEDEADYYC ALWYSNLWVF GGGTKLTVLG 

EV QLVQSGAELK KPGASVKVSC KASGYTFTDY YMKWVRQAPGQGLEWIGDII PSNGATFYNQ KFKGRVTITV DKSTSTAYMELSSLRSEDTA VYYCARSHLL RASWFAYWGQ GTLVTVSS GG CGGGKVAALK EKVAALKEKV AALKEKVAAL KE

Residues 1-110 of the second polypeptide chain of CD123-WT correspond tothe VL Domain of CD3 mAb 1 (SEQ ID NO:56). Residues 111-118 (doubleunderlined) of the second polypeptide chain of CD123-WT correspond toLinker 1 (GGGSGGGG; SEQ ID NO:16). Residues 119-238 of the secondpolypeptide chain of CD123-WT correspond to the VH Domain of CD123 mAb 1(SEQ ID NO:162). Residues 239-244 (underlined) of the second polypeptidechain correspond to Linker 2 (GGCGGG; SEQ ID NO:17). Residues 245-272 ofthe second polypeptide chain of CD123-WT correspond to theheterodimer-promoting “K-coil” (KVAALKE-KVAALKE-KVAALKE-KVAALKE; SEQ IDNO:30).

The third polypeptide chain of CD123-WT has the amino acid sequence ofSEQ ID NO:176:

DKTHTCPPCP APEAAGGPSV FLFPPKPKDT LMISRTPEVTCVVVDVSHED PEVKFNWYVD GVEVHNAKTK PREEQYNSTYRVVSVLTVLH QDWLNGKEYK CKVSNKALPA PIEKTISKAKGQPREPQVYT LPPSREEMTK NQVSLSCAVK GFYPSDIAVEWESNGQPENN YKTTPPVLDS DGSFFLVSKL TVDKSRWQQGNVFSCSVMHE ALHNRYTQKS LSLSPGK

Residues 1-10 of the third polypeptide chain of CD123-WT correspond tothe Linker DKTHTCPPCP (SEQ ID NO:40). Residues 10-227 of the thirdpolypeptide chain of CD123-WT correspond to the IgG1 “hole-bearing”CH2-CH3 Domain (SEQ ID NO:50).

As will be recognized, the third polypeptide chain of CD123-WT does notcontain any Epitope-Binding Domains and may thus be employed in variousDA×CD3 Binding Molecules having such DART-B-type structure.

2. Second Illustrative DART-B-Type Diabody CD123-M1 (CD123×CD3 mAb 1 M1)

A second illustrative DART-B-type diabody is similar to theabove-described CD123-WT diabody, but contains the VH Domain of CD3 mAb1 M1 and is designated “CD123-M1”. As indicated above, CD3 mAb 1 M1 is alow affinity variant of CD3 mAb 1, (also referred to as “CD3 mAb 1Low”). As also indicated above, the VL Domain of CD3 mAb 1 M1 has thesame amino acid sequence as the VL Domain of CD3 mAb 1.

Thus, the second illustrative DART-B-type diabody (CD123-M1) has a firstpolypeptide chain that has the amino acid sequence (SEQ ID NO:177):

DFVMTQSPDS LAVSLGERVT MSCKSSQSLL NSGNQKNYLTWYQQKPGQPP KLLIYWASTR ESGVPDRFSG SGSGTDFTLTISSLQAEDVA VYYCQNDYSY PYTFGQGTKL EIK 

EVQLVESGG GLVQPGGSLR LSCAASGFTF STYAMNWVRQAPGKGLEWVG RIRSKYNNYA TYYADSVK 

R FTISRDDSKN SLYLQMNSLK TEDTAVYYCV RHGNFGNSYV TWFAYWGQGT LVTVSS GGCG  GG EVAALEKE VAALEKEVAA LEKEVAALEK GGGD KTHTCP   PCPAPEAAGG PSVFLFPPKP KDTLMISRTPEVTCVVVDVS HEDPEVKFNW YVDGVEVHNA KTKPREEQYNSTYRVVSVLT VLHQDWLNGK EYKCKVSNKA LPAPIEKTISKAKGQPREPQ VYTLPPSREE MTKNQVSLWC LVKGFYPSDIAVEWESNGQP ENNYKTIPPV LDSDGSFFLY SKLTVDKSRWQQGNVFSCSV MHEALHNHYT QKSLSLSPCK

Residues 1-113 of the first polypeptide chain of CD123-M1 correspond tothe VL Domain of CD123 mAb 1 (SEQ ID NO:163). Residues 114-121 (doubleunderlined) of the first polypeptide chain of CD123-M1 correspond toLinker 1 (GGGSGGGG; SEQ ID NO:16). Residues 122-246 of the firstpolypeptide chain of CD123-M1 correspond to the VH Domain of CD3 mAb 1M1 (SEQ ID NO:55), wherein Kabat position 65 (double underlined) isaspartate (D). Residues 247-252 (underlined) of the first polypeptidechain of CD123-M1 correspond to a Linker 2 (GGCGGG; SEQ ID NO:17).Residues 253-280 of the first polypeptide chain of CD123-M1 correspondto the heterodimer-promoting “E-coil” (EVAALEK-EVAALEK-EVAALEK-EVAALEK;SEQ ID NO:29). Residues 281-283 of the first polypeptide chain ofCD123-M1 correspond to a GGG Linker. Residues 284-293 (underlined) ofthe first polypeptide chain of CD123-M1 correspond to the LinkerDKTHTCPPCP (SEQ ID NO:40). Residues 294-510 of the first polypeptidechain of CD123-M1 correspond to the IgG1 “knob-bearing” CH2-CH3 Domain(SEQ ID NO:48).

Since the VL Domain of CD3 mAb 1 M1 is the same as that of CD3 mAb 1,the amino acid sequence of the second polypeptide chain of CD123-M1 isthe same as that of the second polypeptide chain of the CD123-WT diabody(i.e., SEQ ID NO:175). Similarly, the amino acid sequence of the thirdpolypeptide chain of CD123-M1 is the same as that of the thirdpolypeptide chain of the CD123-WT diabody (i.e., SEQ ID NO:176).

3. Third Illustrative DART-B-Type Diabody CD123-M2 (CD123×CD3 mAb 1 M2)

A third illustrative DART-B-type diabody is similar to theabove-described CD123-M1 diabody, but contains the VH Domain of CD3 mAb1 M2 and is designated “CD123-M2”. As indicated above, CD3 mAb 1 M2 hasa faster off-rate than CD3 mAb 1, and is thus also referred to as “CD3mAb 1 Fast.” As also indicated above, the VL Domain of CD3 mAb 1 M2 hasthe same amino acid sequence as the VL Domain of CD3 mAb 1.

Thus, the third illustrative DART-B-type diabody (CD123-M2) has a firstpolypeptide chain that has the amino acid sequence (SEQ ID NO:178):

DFVMTQSPDS LAVSLGERVT MSCKSSQSLL NSGNQKNYLTWYQQKPGQPP KLLIYWASTR ESGVPDRFSG SGSGTDFTLTISSLQAEDVA VYYCQNDYSY PYTFGQGTKL EIK 

EVQLVESGG GLVQPGGSLR LSCAASGFTF STYAMNWVRQAPGKGLEWVG RIRSKYNNYA TYYADSVK 

R FTISRDDSKN SLYLQMNSLK TEDTAVYYCV RHKNFGNSYV TWFAYWGQGT LVTVSS GGCG  GG EVAALEKE VAALEKEVAA LEKEVAALEK GGG DKTHTCP   PCPAPEAAGG PSVFLFPPKP KDTLMISRTPEVTCVVVDVS HEDPEVKFNW YVDGVEVHNA KTKPREEQYNSTYRVVSVLT VLHQDWLNGK EYKCKVSNKA LPAPIEKTISKAKGQPREPQ VYTLPPSREE MTKNQVSLWC LVKGFYPSDIAVEWESNGQP ENNYKTTPPV LDSDGSFFLY SKLTVDKSRWQQGNVFSCSV MHEALHNHYT QKSLSLSPGK

Residues 1-113 of the first polypeptide chain of CD123-M2 correspond tothe VL Domain of CD123 mAb 1 (SEQ ID NO:163). Residues 114-121 (doubleunderlined) of the first polypeptide chain of CD123-M2 correspond toLinker 1 (GGGSGGGG; SEQ ID NO:16). Residues 122-246 of the firstpolypeptide chain of CD123-M2 correspond to the VH Domain of CD3 mAb 1M2 (SEQ ID NO:59), wherein Kabat position 65 (double underlined) isaspartate (D). Residues 247-252 (underlined) of the first polypeptidechain of CD123-M2 correspond to a Linker 2 (GGCGGG; SEQ ID NO:17).Residues 253-280 of the first polypeptide chain of CD123-M2 correspondto the heterodimer-promoting “E-coil” (EVAALEK-EVAALEK-EVAALEK-EVAALEK;SEQ ID NO:29). Residues 281-283 of the first polypeptide chain ofCD123-M2 correspond to a GGG Linker. Residues 284-293 (underlined) ofthe first polypeptide chain of CD123-M2 correspond to the LinkerDKTHTCPPCP (SEQ ID NO:40). Residues 294-510 of the first polypeptidechain of CD123-M2 correspond to the IgG1 “knob-bearing” CH2-CH3 Domain(SEQ ID NO:48).

Since the VL Domain of CD3 mAb 1 M2 is the same as that of CD3 mAb 1,the amino acid sequence of the second polypeptide chain of CD123-M2 isthe same as that of the second polypeptide chain of the CD123-WT diabody(i.e., SEQ ID NO:175). Similarly, the amino acid sequence of the thirdpolypeptide chain of CD123-M2 is the same as that of the thirdpolypeptide chain of the CD123-WT diabody (i.e., SEQ ID NO:176).

4. Fourth Illustrative DART-B-Type Diabody CD123-M18 (CD123×CD3 mAb 1M18)

A fourth illustrative DART-B-type diabody is similar to theabove-described CD123-M2 diabody, but contains the VH Domain of CD3 mAb1 M18 and is designated “CD123-M18”. As indicated above, the VL Domainof CD3 mAb 1 M18 has the same amino acid sequence as the VL Domain ofCD3 mAb 1.

Thus, the fourth illustrative DART-B-type diabody (CD123-M18) has afirst polypeptide chain that has the amino acid sequence (SEQ IDNO:179):

DFVMTQSPDS LAVSLGERVT MSCKSSQSLL NSGNQKNYLTWYQQKPGQPP KLLIYWASTR ESGVPDRFSG SGSGTDFTLTISSLQAEDVA VYYCQNDYSY PYTFGQGTKL EIK 

EVQLVESGG GLVQPGGSLR LSCAASGFTF STYGMNWVRQAPGKGLEWVG RIRSKYNNYA TYYADSVK 

R FTISRDDSKN SLYLQMNSLK TEDTAVYYCV RHGNFGNSYV SWFAYWGQGT LVTVSS GGCG  GG EVAALEKE VAALEKEVAA LEKEVAALEK GGG DKTHTCP   PCPAPEAAGG PSVFLFPPKP KDTLMISRTPEVTCVVVDVS HEDPEVKFNW YVDGVEVHNA KTKPREEQYNSTYRVVSVLT VLHQDWLNGK EYKCKVSNKA LPAPIEKTISKAKGQPREPQ VYTLPPSREE MTKNQVSLWC LVKGFYPSDIAVEWESNGQP ENNYKTTPPV LDSDGSFFLY SKLTVDKSRWQQGNVFSCSV MHEALHNHYT QKSLSLSPGK

Residues 1-113 of the first polypeptide chain of CD123-M18 correspond tothe VL Domain of CD123 mAb 1 (SEQ ID NO:163). Residues 114-121 (doubleunderlined) of the first polypeptide chain of CD123-M18 correspond toLinker 1 (GGGSGGGG; SEQ ID NO:16). Residues 122-246 of the firstpolypeptide chain of CD123-M18 correspond to the VH Domain of CD3 mAb 1M18 (SEQ ID NO:98), wherein Kabat position 65 (double underlined) isaspartate (D). Residues 247-252 (underlined) of the first polypeptidechain of CD123-M18 correspond to a Linker 2 (GGCGGG; SEQ ID NO:17).Residues 253-280 of the first polypeptide chain of CD123-M18 correspondto the heterodimer-promoting “E-coil” (EVAALEK-EVAALEK-EVAALEK-EVAALEK;SEQ ID NO:29). Residues 281-283 of the first polypeptide chain ofCD123-M18 correspond to a GGG Linker. Residues 284-293 (underlined) ofthe first polypeptide chain of CD123-M18 correspond to the LinkerDKTHTCPPCP (SEQ ID NO:40). Residues 294-510 of the first polypeptidechain of CD123-M18 correspond to the IgG1 “knob-bearing” CH2-CH3 Domain(SEQ ID NO:48).

Since the VL Domain of CD3 mAb 1 M18 is the same as that of CD3 mAb 1,the amino acid sequence of the second polypeptide chain of CD123-M18 isthe same as that of the second polypeptide chain of the CD123-WT diabody(i.e., SEQ ID NO:175). Similarly, the amino acid sequence of the thirdpolypeptide chain of CD123-M18 is the same as that of the thirdpolypeptide chain of the CD123-WT diabody (i.e., SEQ ID NO:176).

5. Fifth Illustrative DART-B-Type Diabody CD123-M13 (CD123×CD3 mAb 1M13)

A fifth illustrative DART-B-type diabody is similar to theabove-described CD123-WT diabody, but contains the VH Domain of CD3 mAb1 M13 and is designated “CD123-M13”. As indicated above, the VL Domainof CD3 mAb 1 M13 has the same amino acid sequence as the VL Domain ofCD3 mAb 1.

Thus, the fifth illustrative DART-B-type diabody (CD123-M13) has a firstpolypeptide chain that has the amino acid sequence (SEQ ID NO:198):

DFVMTQSPDS LAVSLGERVT MSCKSSQSLL NSGNQKNYLTWYQQKPGQPP KLLIYWASTR ESGVPDRFSG SGSGTDFTLTISSLQAEDVA VYYCQNDYSY PYTFOQGTKL EIK 

EVQLVESGG GLVQPGGSLR LSCAASGFTF STYAMNWVRQAPGKGLEWVG RIRSKYNNYA TYYADSVK 

R FTISRDDSKN SLYLQMNSLK TEDTAVYYCV RHGNFGNSYV SWFAEWGQGT LVTVSS GGCG  GG EVAALEKE VAALEKEVAA LEKEVAALEK GGG DKTHTCP   PCPAPEAAGG PSVFLFPPKP KDTLMISRTPEVTCVVVDVS HEDPEVKFNW YVDGVEVHNA KTKPREEQYNSTYRVVSVLT VLHQDWLNGK EYKCKVSNKA LPAPIEKTISKAKGQPREPQ VYTLPPSREE MTKNQVSLWC LVKGFYPSDIAVEWESNGQP ENNYKTTPPV LDSDGSFFLY SKLTVDKSRWQQGNVFSCSV MHEALHNHYT QKSLSLSPGK

Residues 1-113 of the first polypeptide chain of CD123-M13 correspond tothe VL Domain of CD123 mAb 1 (SEQ ID NO:163). Residues 114-121 (doubleunderlined) of the first polypeptide chain of CD123-M13 correspond toLinker 1 (GGGSGGGG; SEQ ID NO:16). Residues 122-246 of the firstpolypeptide chain of CD123-M13 correspond to the VH Domain of CD3 mAb 1M13 (SEQ ID NO:88), wherein Kabat position 65 (double underlined) isaspartate (D). Residues 247-252 (underlined) of the first polypeptidechain of CD123-M1 correspond to a Linker 2 (GGCGGG; SEQ ID NO:17).Residues 253-280 of the first polypeptide chain of CD123-M13 correspondto the heterodimer-promoting “E-coil” (EVAALEK-EVAALEK-EVAALEK-EVAALEK;SEQ ID NO:29). Residues 281-283 of the first polypeptide chain ofCD123-M13 correspond to a GGG Linker. Residues 284-293 (underlined) ofthe first polypeptide chain of CD123-M13 correspond to the LinkerDKTHTCPPCP (SEQ ID NO:40). Residues 294-510 of the first polypeptidechain of CD123-M13 correspond to the IgG1 “knob-bearing” CH2-CH3 Domain(SEQ ID NO:48).

Since the VL Domain of CD3 mAb 1 M13 is the same as that of CD3 mAb 1,the amino acid sequence of the second polypeptide chain of CD123-M13 isthe same as that of the second polypeptide chain of the CD123-WT diabody(i.e., SEQ ID NO:175). Similarly, the amino acid sequence of the thirdpolypeptide chain of CD123-M1 is the same as that of the thirdpolypeptide chain of the CD123-WT diabody (i.e., SEQ ID NO:176).

6. Sixth Illustrative DART-B-Type Diabody CD123-M17 (CD123×CD3 mAb 1M17)

A sixth illustrative DART-B-type diabody is similar to theabove-described CD123-WT diabody, but contains the VH Domain of CD3 mAb1 M17 and is designated “CD123-M17”. As indicated above, the VL Domainof CD3 mAb 1 M17 has the same amino acid sequence as the VL Domain ofCD3 mAb 1.

Thus, the sixth illustrative DART-B-type diabody (CD123-M17) has a firstpolypeptide chain that has the amino acid sequence (SEQ ID NO:199):

DFVMTQSPDS LAVSLGERVT MSCKSSQSLL NSGNQKNYLTWYQQKPGQPP KLLIYWASTR ESGVPDRFSG SGSGTDFTLTISSLQAEDVA VYYCQNDYSY PYTFGQGTKL EIK 

EVQLVESGG GLVQPGGSLR LSCAASGFTF STTAMNWVRQAPGKGLEWVG RIRSKYNNYA TYYADSVK 

R FTISRDDSKN SLYLQMNSLK TEDTAVYYCV RHGNFGNSYV SWFAYWGQGT LVTVSS GGCG  GG EVAALEKE VAALEKEVAA LEKEVAALEK GGG DKTHTCP   PCPAPEAAGG PSVFLFPPKP KDTLMISRTPEVTCVVVDVS HEDPEVKFNW YVDGVEVHNA KTKPREEQYNSTYRVVSVLT VLHQDWLNGK EYKCKVSNKA LPAPIEKTISKAKGQPREPQ VYTLPPSREE MTKNQVSLWC LVKGFYPSDIAVEWESNGQP ENNYKTTPPV LDSDGSFFLY SKLTVDKSRWQQGNVFSCSV MHEALHNHYT QKSLSLSPGK

Residues 1-113 of the first polypeptide chain of CD123-M17 correspond tothe VL Domain of CD123 mAb 1 (SEQ ID NO:163). Residues 114-121 (doubleunderlined) of the first polypeptide chain of CD123-M17 correspond toLinker 1 (GGGSGGGG; SEQ ID NO:16). Residues 122-246 of the firstpolypeptide chain of CD123-M17 correspond to the VH Domain of CD3 mAb 1M17 (SEQ ID NO:96), wherein Kabat position 65 (double underlined) isaspartate (D). Residues 247-252 (underlined) of the first polypeptidechain of CD123-M17 correspond to a Linker 2 (GGCGGG; SEQ ID NO:17).Residues 253-280 of the first polypeptide chain of CD123-M17 correspondto the heterodimer-promoting “E-coil” (EVAALEK-EVAALEK-EVAALEK-EVAALEK;SEQ ID NO:29). Residues 281-283 of the first polypeptide chain ofCD123-M17 correspond to a GGG Linker. Residues 284-293 (underlined) ofthe first polypeptide chain of CD123-M17 correspond to the LinkerDKTHTCPPCP (SEQ ID NO:40). Residues 294-510 of the first polypeptidechain of CD123-M17 correspond to the IgG1 “knob-bearing” CH2-CH3 Domain(SEQ ID NO:48).

Since the VL Domain of CD3 mAb 1 M17 is the same as that of CD3 mAb 1,the amino acid sequence of the second polypeptide chain of CD123-M17 isthe same as that of the second polypeptide chain of the CD123-WT diabody(i.e., SEQ ID NO:175). Similarly, the amino acid sequence of the thirdpolypeptide chain of CD123-M17 is the same as that of the thirdpolypeptide chain of the CD123-WT diabody (i.e., SEQ ID NO:176).

7. Seventh Illustrative DART-B-Type Diabody CD123-M19 (CD123×CD3 mAb 1M19)

A seventh illustrative DART-B-type diabody is similar to theabove-described CD123-WT diabody, but contains the VH Domain of CD3 mAb1 M19 and is designated “CD123-M19”. As indicated above, the VL Domainof CD3 mAb 1 M19 has the same amino acid sequence as the VL Domain ofCD3 mAb 1.

Thus, the seventh illustrative DART-B-type diabody (CD123-M19) has afirst polypeptide chain that has the amino acid sequence (SEQ IDNO:200):

DFVMTQSPDS LAVSLGERVT MSCKSSQSLL NSGNQKNYLTWYQQKPGQPP KLLIYWASTR ESGVPDRFSG SGSGTDFTLTISSLQAEDVA VYYCQNDYSY PYTFGQGTKL EIK 

EVQLVESGG GLVQPGGSLR LSCAASGFTF STYAMNWVRQAPGKGLEWVG RIRSKYNNYA TYYADSVK 

R FTISRDDSKN SLYLQMNSLK TEDTAVYYCV RHKNIGNSYV SWFAYWGQGT LVTVSS GGCG  GG EVAALEKE VAALEKEVAA LEKEVAALEK GGG DKTHTCP   PCPAPEAAGG PSVFLFPPKP KDTLMISRTPEVTCVVVDVS HEDPEVKFNW YVDGVEVHNA KTKPREEQYNSTYRVVSVLT VLHQDWLNGK EYKCKVSNKA LPAPIEKTISKAKGQPREPQ VYTLPPSREE MTKNQVSLWC LVKGFYPSDIAVEWESNGQP ENNYKTTPPV LDSDGSFFLY SKLTVDKSRWQQGNVFSCSV MHEALHNHYT QKSLSLSPGK

Residues 1-113 of the first polypeptide chain of CD123-M19 correspond tothe VL Domain of CD123 mAb 1 (SEQ ID NO:163). Residues 114-121 (doubleunderlined) of the first polypeptide chain of CD123-M19 correspond toLinker 1 (GGGSGGGG; SEQ ID NO:16). Residues 122-246 of the firstpolypeptide chain of CD123-M19 correspond to the VH Domain of CD3 mAb 1M19 (SEQ ID NO:100), wherein Kabat position 65 (double underlined) isaspartate (D). Residues 247-252 (underlined) of the first polypeptidechain of CD123-M19 correspond to a Linker 2 (GGCGGG; SEQ ID NO:17).Residues 253-280 of the first polypeptide chain of CD123-M19 correspondto the heterodimer-promoting “E-coil” (EVAALEK-EVAALEK-EVAALEK-EVAALEK;SEQ ID NO:29). Residues 281-283 of the first polypeptide chain ofCD123-M19 correspond to a GGG Linker. Residues 284-293 (underlined) ofthe first polypeptide chain of CD123-M1 correspond to the LinkerDKTHTCPPCP (SEQ ID NO:40). Residues 294-510 of the first polypeptidechain of CD123-M19 correspond to the IgG1 “knob-bearing” CH2-CH3 Domain(SEQ ID NO:48).

Since the VL Domain of CD3 mAb 1 M19 is the same as that of CD3 mAb 1,the amino acid sequence of the second polypeptide chain of CD123-M19 isthe same as that of the second polypeptide chain of the CD123-WT diabody(i.e., SEQ ID NO:175). Similarly, the amino acid sequence of the thirdpolypeptide chain of CD123-M1 is the same as that of the thirdpolypeptide chain of the CD123-WT diabody (i.e., SEQ ID NO:176).

8. Eighth Illustrative DART-B-Type Diabody 5T4-WT (5T4×CD3 mAb 1)

An eighth illustrative DART-B-type diabody is similar to theabove-described CD123-M18 diabody, but comprises a 5T4 Binding Domain inlieu of the CD123 Binding Domain of the CD123-M18 diabody. Additionally,the eighth illustrative DART-B-type diabody contains the VH Domain ofCD3 mAb 1. This eighth illustrative DART-B-type diabody is designated“5T4-WT”.

Thus, the eighth illustrative DART-B-type diabody (5T4-WT) has a firstpolypeptide chain that has the amino acid sequence (SEQ ID NO:180):

DIQMTQSPSS LSASVGDRVT ITCRASQGIS NYLAWFQQKPGKAPKSLIYR ANRLQSGVPS RFSGSGSGTD FTLTISSLQPEDVATYYCLQ YDDFPWTFGQ GTKLEIK 

 

EVQLV ESGGGLVQPG GSLRLSCAAS GFTFSTYAMN WVRQAPGKGLEWVGRIRSKY NNYATYYADS VK 

RFTISRD DSKNSLYLQM NSLKTEDTAV YYCVRHGNFG NSYVSWFAYW GQGTLVTVSS GGCGGGEVAA LEKEVAALEK EVAALEKEVA ALEKGGG DKT HTCPPCPAPE AAGGPSVFLF PPKPKDILMI SRTPEVTCVVVDVSEEDPEV KFNWYVDGVE VHNAKTKPRE EQYNSTYRVVSVLTVLHQDW LNGKEYKCKV SNKALPAPIE KTISKAKGQPREPQVYTLPP SREEMTKNQV SLWCLVKGFY PSDIAVEWESNGQPENNYKT TPPVLDSDGS FFLYSKLTVD KSRWQQGNVF SCSVMHEALH NHYTQKSLSL SPGK

Residues 1-107 of the first polypeptide chain of 5T4-WT correspond tothe VL Domain of 5T4 mAb 1 (SEQ ID NO:157). Residues 108-115 (doubleunderlined) of the first polypeptide chain of 5T4-WT correspond toLinker 1 (GGGSGGGG; SEQ ID NO:16). Residues 116-240 of the firstpolypeptide chain of 5T4-WT correspond to the VH Domain of CD3 mAb 1(SEQ ID NO:55), wherein Kabat position 65 (double underlined) is glycine(G). Residues 241-246 (underlined) of the first polypeptide chain of5T4-WT correspond to a Linker 2 (GGCGGG; SEQ ID NO:17). Residues 247-274of the first polypeptide chain of 5T4-WT correspond to theheterodimer-promoting “E-coil” (EVAALEK-EVAALEK-EVAALEK-EVAALEK; SEQ IDNO:29). Residues 275-277 of the first polypeptide chain of 5T4-WTcorrespond to a GGG Linker. Residues 278-287 (underlined) of the firstpolypeptide chain of 5T4-WT correspond to the Linker DKTHTCPPCP (SEQ IDNO:40). Residues 288-504 of the first polypeptide chain of 5T4-WTcorrespond to the IgG1 “knob-bearing” CH2-CH3 Domain (SEQ ID NO:48).

The second polypeptide chain of 5T4-WT has the amino acid sequence (SEQID NO:181):

QAVVTQEPSL TVSPGGTVTL TGRSSTGAVT TSNYANWVQQKPGQAPRGLI GGTNKRAPWT PARFSGSLLG GKAALTITGAQAEDEADYYC ALWYSNLWVF GGGTKLTVLG 

QV QLVQSGAEVK KPGASVKVSC KASGYTFTSF WMHWVRQAPGQGLEWMGRID PNRGGTEYNE KAKSRVTMTA DKSTSTAYMELSSLRSEDTA VYYCAGGNPY YPMDYWGQGT TVTVSS GGCG GGKVAALKEK VAALKEKVAA LKEKVAALKE

Residues 1-110 of the second polypeptide chain of 5T4-WT correspond tothe VL Domain of CD3 mAb 1 (SEQ ID NO:56). Residues 111-118 (doubleunderlined) of the second polypeptide chain of 5T4-WT correspond toLinker 1 (GGGSGGGG; SEQ ID NO:16). Residues 119-236 of the secondpolypeptide chain of 5T4-WT correspond to the VH Domain of 5T4 mAb 1(SEQ ID NO:156). Residues 237-242 (underlined) of the second polypeptidechain of 5T4-WT correspond to a Linker 2 (GGCGGG; SEQ ID NO:17).Residues 243-280 of the second polypeptide chain of 5T4-WT correspond tothe heterodimer-promoting “K-coil” (KVAALKE-KVAALKE-KVAALKE-KVAALKE; SEQID NO:30).

The third polypeptide chain of 5T4-WT has the same amino acid sequenceas the third polypeptide chain of the CD123-WT diabody (i.e., SEQ IDNO:176).

9. Ninth Illustrative DART-B-Type Diabody 5T4-M1 (5T4×CD3 mAb 1 M1)

A ninth illustrative DART-B-type diabody is similar to theabove-described 5T4-WT diabody, but comprises the VH Domain of CD3 mAb 1M1 and is designated “5T4-M1.”

Thus, the ninth illustrative DART-B-type diabody (5T4-M1) has a firstpolypeptide chain that has the amino acid sequence (SEQ ID NO:182):

DIQMTQSPSS LSASVGDRVT ITCRASQGIS NYLAWFQQKPGKAPKSLIYR ANRLQSGVPS RFSGSGSGTD FTLTISSLQPEDVATYYCLQ YDDFPWTFGQ GTKLEIK 

 

EVQLV ESGGGLVQPG GSLRLSCAAS GFTFSTYAMN WVRQAPGKGLEWVGRIRSKY NNYATYYADS VK 

RFTISRD DSKNSLYLQM NSLKTEDTAV YYCVRHGNFG NSYVTWFAYW GQGTLVTVSS GGCGGGEVAA LEKEVAALEK EVAALEKEVA ALEKGGG DKT HTCPPCPAPE AAGGPSVFLF PPKPKDILMI SRTPEVTCVVVDVSHEDPEV KFNWYVDGVE VHNAKTKPRE EQYNSTYRVVSVLTVLHQDW LNGKEYKCKV SNKALPAPIE KTISKAKGQPREPQVYTLPP SREEMTKNQV SLWCLVKGFY PSDIAVEWESNGQPENNYKT TPPVLDSDGS FFLYSKLTVD KSRWQQGNVF SCSVMHEALH NHYTQKSLSL SPGK

Residues 1-107 of the first polypeptide chain of 5T4-M1 correspond tothe VL Domain of 5T4 mAb 1 (SEQ ID NO:157). Residues 108-115 (doubleunderlined) of the first polypeptide chain of 5T4-M1 correspond toLinker 1 (GGGSGGGG; SEQ ID NO:16). Residues 116-240 of the firstpolypeptide chain of 5T4-M1 correspond to the VH Domain of CD3 mAb 1 M1(SEQ ID NO:64), wherein Kabat position 65 (double underlined) isaspartate (D). Residues 241-246 (underlined) of the first polypeptidechain of 5T4-M1 correspond to a Linker 2 (GGCGGG; SEQ ID NO:17).Residues 247-274 of the first polypeptide chain of 5T4-M1 correspond tothe heterodimer-promoting “E-coil” (EVAALEK-EVAALEK-EVAALEK-EVAALEK; SEQID NO:29). Residues 275-277 of the first polypeptide chain of 5T4-M1correspond to a GGG Linker. Residues 278-287 (underlined) of the firstpolypeptide chain of 5T4-M1 correspond to the Linker DKTHTCPPCP (SEQ IDNO:40). Residues 288-504 of the first polypeptide chain of 5T4-M1correspond to the IgG1 “knob-bearing” CH2-CH3 Domain (SEQ ID NO:48).

Since the VL Domain of CD3 mAb 1 M1 is the same as that of CD3 mAb 1,the amino acid sequence of the second polypeptide chain of 5T4-M1 is thesame as that of the second polypeptide chain of the 5T4-WT diabody(i.e., SEQ ID NO:181). Similarly, the amino acid sequence of the thirdpolypeptide chain of 5T4-M1 is the same as that of the third polypeptidechain of the CD123-WT diabody (i.e., SEQ ID NO:176).

10. Tenth Illustrative DART-B-Type Diabody 5T4-M2 (5T4×CD3 mAb 1 M2)

A tenth illustrative DART-B-type diabody is similar to theabove-described 5T4-M1 diabody, but comprises the VH Domain of CD3 mAb 1M2 and is designated “5T4-M2”.

Thus, the tenth illustrative DART-B-type diabody (5T4-M2) has a firstpolypeptide chain that has the amino acid sequence (SEQ ID NO:183):

DIQMTQSPSS LSASVGDRVT ITCRASQGIS NYLAWFQQKPGKAPKSLIYR ANRLQSGVPS RFSGSGSGTD FTLTISSLQPEDVATYYCLQ YDDFPWTFGQ GTKLEIK 

 

EVQLV ESGGGLVQPG GSLRLSCAAS GFTFSTYAMN WVRQAPGKGLEWVGRIRSKY NNYATYYADS VK 

RFTISRD DSKNSLYLQM NSLKTEDTAV YYCVRHKNFG NSYVTWFAYW GQGTLVTVSS GGCGGGEVAA LEKEVAALEK EVAALEKEVA ALEKGGG DKT HTCPPCPAPE AAGGPSVFLF PPKPKDTLMI SRTPEVTCVVVDVSHEDPEV KFNWYVDGVE VHNAKTKPRE EQYNSTYRVVSVLTVLHQDW LNGKEYKCKV SNKALPAPIE KTISKAKGQPREPQVYTLPP SREEMTKNQV SLWCLVKGFY PSDIAVEWESNGQPENNYKT TPPVLDSDGS FFLYSKLTVD KSRWQQGNVF SCSVMHEALH NHYTQKSLSL SPGK

Residues 1-107 of the first polypeptide chain of 5T4-M2 correspond tothe VL Domain of 5T4 mAb 1 (SEQ ID NO:157). Residues 108-115 (doubleunderlined) of the first polypeptide chain of 5T4-M2 correspond toLinker 1 (GGGSGGGG; SEQ ID NO:16). Residues 116-240 of the firstpolypeptide chain of 5T4-M2 correspond to the VH Domain of CD3 mAb 1 M2(SEQ ID NO:66), wherein Kabat position 65 (double underlined) isaspartate (D). Residues 241-246 (underlined) of the first polypeptidechain of 5T4-M2 correspond to a Linker 2 (GGCGGG; SEQ ID NO:17).Residues 247-274 of the first polypeptide chain of 5T4-M2 correspond tothe heterodimer-promoting “E-coil” (EVAALEK-EVAALEK-EVAALEK-EVAALEK; SEQID NO:29). Residues 275-277 of the first polypeptide chain of 5T4-M2correspond to a GGG Linker. Residues 278-287 (underlined) of the firstpolypeptide chain of 5T4-M2 correspond to the Linker DKTHTCPPCP (SEQ IDNO:40). Residues 288-504 of the first polypeptide chain of 5T4-M2correspond to the IgG1 “knob-bearing” CH2-CH3 Domain (SEQ ID NO:48).

Since the VL Domain of CD3 mAb 1 M2 is the same as that of CD3 mAb 1,the amino acid sequence of the second polypeptide chain of 5T4-M2 is thesame as that of the second polypeptide chain of the 5T4-WT diabody(i.e., SEQ ID NO:181). Similarly, the amino acid sequence of the thirdpolypeptide chain of 5T4-M2 is the same as that of the third polypeptidechain of the CD123-WT diabody (i.e., SEQ ID NO:176).

11. Eleventh Illustrative DART-B-Type Diabody 5T4-M18 (5T4×CD3 mAb 1M18)

An eleventh illustrative DART-B-type diabody is similar to theabove-described 5T4-WT diabody, but comprises the VH Domain of CD3 mAb 1M18 and is designated “5T4-M18”.

Thus, the eleventh illustrative DART-B-type diabody (5T4-M18) has afirst polypeptide chain that has the amino acid sequence (SEQ IDNO:184):

DIQMTQSPSS LSASVGDRVT ITCRASQGIS NYLAWFQQKPGKAPKSLIYR ANRLQSGVPS RFSGSGSGTD FTLTISSLQPEDVATYYCLQ YDDFPWTFGQ GTKLEIK 

 

EVQLV ESGGGLVQPG GSLRLSCAAS GFTFSTYGMN WVRQAPGKGLEWVGRIRSKY NNYATYYADS VK 

RFTISRD DSKNSLYLQM NSLKTEDTAV YYCVRHGNFG NSYVSWFAYW GQGTLVTVSS GGCGGGEVAA LEKEVAALEK EVAALEKEVA ALEKGGG DKT HTCPPCPAPE AAGGPSVFLF PPKPKDTLMI SRTPEVTCVVVDVSHEDPEV KFNWYVDGVE VHNAKTKPRE EQYNSTYRVVSVLTVLHQDW LNGKEYKCKV SNKALPAPIE KTISKAKGQPREPQVYTLPP SREEMTKNQV SLWCLVKGFY PSDIAVEWESNGQPENNYKT TPPVLDSDGS FFLYSKLTVD KSRWQQGNVF SCSVMHEALH NHYTQKSLSL SPGK

Residues 1-107 of the first polypeptide chain of 5T4-M18 correspond tothe VL Domain of 5T4 mAb 1 (SEQ ID NO:157). Residues 108-115 (doubleunderlined) of the first polypeptide chain of 5T4-M18 correspond toLinker 1 (GGGSGGGG; SEQ ID NO:16). Residues 116-240 of the firstpolypeptide chain of 5T4-M18 correspond to the VH Domain of CD3 mAb 1M18 (SEQ ID NO:98), wherein Kabat position 65 (double underlined) isaspartate (D). Residues 241-246 (underlined) of the first polypeptidechain of 5T4-M18 correspond to a Linker 2 (GGCGGG; SEQ ID NO:17).Residues 247-274 of the first polypeptide chain of 5T4-M18 correspond tothe heterodimer-promoting “E-coil” (EVAALEK-EVAALEK-EVAALEK-EVAALEK; SEQID NO:29). Residues 275-277 of the first polypeptide chain of 5T4-M18correspond to a GGG Linker. Residues 278-287 (underlined) of the firstpolypeptide chain of 5T4-M18 correspond to the Linker DKTHTCPPCP (SEQ IDNO:40). Residues 288-504 of the first polypeptide chain of 5T4-M18correspond to the IgG1 “knob-bearing” CH2-CH3 Domain (SEQ ID NO:48).

Since the VL Domain of CD3 mAb 1 M18 is the same as that of CD3 mAb 1,the amino acid sequence of the second polypeptide chain of 5T4-M18 isthe same as that of the second polypeptide chain of the 5T4-WT diabody(i.e., SEQ ID NO:181). Similarly, the amino acid sequence of the thirdpolypeptide chain of 5T4-M18 is the same as that of the thirdpolypeptide chain of the CD123-WT diabody (i.e., SEQ ID NO:176).

12. Twelfth Illustrative DART-B-Type Diabody HIV-WT (HIV×CD3 mAb 1)

A twelfth illustrative DART-B-type diabody is similar to theabove-described CD123-WT diabody, but comprises the HIV Binding Domainof the anti-HIV antibody A32 in lieu of the CD123 Binding Domain of theCD123-WT diabody. This twelfth illustrative DART-B-type diabody isdesignated “HIV-WT”.

Thus, the twelfth illustrative DART-B-type diabody (HIV-WT) has a firstpolypeptide chain that has the amino acid sequence (SEQ ID NO:185):

QSALTQPPSA SGSPGQSVTI SCTGTSSDVG GYNYVSWYQHHPGKAPKLII SEVNNRPSGV PDRFSGSKSG NTASLTVSGLQAEDEAEYYC SSYTDIHNFV FGGGTKLTVL 

EV QLVESGGGLV QPGGSLRLSC AASGFTFSTY AMNWVRQAPGKGLEWVGRIR SKYNNYATYY ADSVK 

RFTI SRDDSKNSLY LQMNSLKTED TAVYYCVRHG NFGNSYVSWF AYWGQGTLVT VSS ASTKGEV AACEKEVAAL EKEVAALEKE VAALEKGGG D KTHTCPPCPA PEAAGGPSVF LFPPKPKDTL MISRTPEVTCVVVDVSEEDP EVKFNWYVDG VEVHNAKTKP REEQYNSTYRVVSVLTVLHQ DWLNGKEYKG KVSNKALPAP IEKTISKAKGQPREPQVYTL PPSREEMTKN QVSLWCLVKG FYPSDIAVEWESNGQPENNY KTTPPVLDSD GSFFLYSKLT VDKSRWQQGN VFSCSVMHEA LHNHYTQKSL SLSPGK

Residues 1-110 of the first polypeptide chain of HIV-WT correspond tothe VL Domain of A32 (SEQ ID NO:169). Residues 111-118 (doubleunderlined) of the first polypeptide chain of HIV-WT correspond toLinker 1 (GGGSGGGG; SEQ ID NO:16). Residues 119-243 of the firstpolypeptide chain of HIV-WT correspond to the VH Domain of CD3 mAb 1(SEQ ID NO:55), wherein Kabat position 65 (double underlined) is glycine(G). Residues 244-248 (underlined) of the first polypeptide chain ofHIV-WT correspond to a Linker 2 (ASTKG; SEQ ID NO:21; singleunderlined). Residues 249-276 of the first polypeptide chain of HIV-WTcorrespond to the heterodimer-promoting “E-coil” (EVAA

EK-EVAALEK-EVAALEK-EVAALEK; SEQ ID NO:31). Residues 277-279 of the firstpolypeptide chain of HIV-WT correspond to a GGG Linker. Residues 280-289(underlined) of the first polypeptide chain of HIV-WT correspond to theLinker DKTHTCPPCP (SEQ ID NO:40; single underlined). Residues 290-506 ofthe first polypeptide chain of HIV-WT correspond to the IgG1“knob-bearing” CH2-CH3 Domain (SEQ ID NO:48).

The second polypeptide chain of HIV-WT has the amino acid sequence (SEQID NO:186):

QAVVTQEPSL TVSPGGTVTL TCRSSTGAVT TSNYANWVQQKPGQAPRGLI GGTNKRAPWT PARFSGSLLG GKAALTITGAQAEDEADYYC ALWYSNLWVF GGGTKLTVLG 

QV QLQESGPGLV KPSQTLSLSC TVSGGSSSSG AHYWSWIRQYPGKGLEWIGY IHYSGNTYYN PSLKSRITIS QHTSENQFSLKLNSVTVADT AVYYCARGTR LRTLRNAFDI WGQGTLVTVS S ASTKGKVAA CKEKVAALKE KVAALKEKVA ALKE

Residues 1-110 of the second polypeptide chain of HIV-WT correspond tothe VL Domain of CD3 mAb 1 (SEQ ID NO:56). Residues 111-118 (doubleunderlined) of the second polypeptide chain of HIV-WT correspond toLinker 1 (GGGSGGGG; SEQ ID NO:16). Residues 119-241 of the secondpolypeptide chain of HIV-WT correspond to the VH Domain of A32 (SEQ IDNO:209 (i.e., SEQ ID NO:168, wherein X is L)). Residues 242-246(underlined) of the second polypeptide chain of HIV-WT correspond to aLinker 2 (ASTKG; SEQ ID NO:21). Residues 247-274 of the secondpolypeptide chain of HIV-WT correspond to the heterodimer-promoting“K-coil” (KVAA

KE-KVAALKE-KVAALKE-KVAALKE; SEQ ID NO:32).

The third polypeptide chain of HIV-WT has the same amino acid sequenceas the third polypeptide chain of the CD123-WT diabody (i.e., SEQ IDNO:176).

13. Thirteenth Illustrative DART-B-Type Diabody HIV-M18 (HIV×CD3 mAb 18)

A thirteenth illustrative DART-B-type diabody is similar to theabove-described HIV-WT diabody, but contains the VH Domain of CD3 mAb 1M18. This illustrative DART-B-type diabody is designated “HIV-M18”.

Thus, the thirteenth illustrative DART-B-type diabody (HIV-M18) has afirst polypeptide chain that has the amino acid sequence (SEQ IDNO:196):

QSALTQPPSA SGSPGQSVTI SCTGTSSDVG GYNYVSWYQH HPGKAPKLII SEVNNRPSGVPDRFSGSKSG NTASLTVSGL QAEDEAEYYC SSYTDIHNFV FGGGTKLTVL 

EV QLVESGGGLV QPGGSLRLSC AASGFTFSTY GMNWVRQAPG KGLEWVGRIR SKYNNYATYYADSVK 

RFTI SRDDSKNSLY LQMNSLKTED TAVYYCVRHG NFGNSYVSWF AYWGQGTLVT VSS ASTKGEV AACEKEVAAL EKEVAALEKE VAALEKGGG D KTHTCPPCP A PEAAGGPSVFLFPPKPKDTL MISRTPEVTC VVVDVSHEDP EVKFNWYVDG VEVHNAKTKP REEQYNSTYRVVSVLTVLHQ DWLNGKEYKC KVSNKALPAP IEKTISKAKG QPREPQVYTL PPSREEMTKNQVSLWCLVKG FYPSDIAVEW ESNGQPENNY KTTPPVLDSD GSFFLYSKLT VDKSRWQQGNVFSCSVMHEA LHNHYTQKSL SLSPGK

Residues 1-110 of the first polypeptide chain of HIV-M18 correspond tothe VL Domain of A32 (SEQ ID NO:169). Residues 111-118 (doubleunderlined) of the first polypeptide chain of HIV-M18 correspond toLinker 1 (GGGSGGGG; SEQ ID NO:16). Residues 119-243 of the firstpolypeptide chain of HIV-M18 correspond to the VH Domain of CD3 mAb 1M18 (SEQ ID NO:55), wherein Kabat position 65 (double underlined) isglycine (G). Residues 244-248 (underlined) of the first polypeptidechain of HIV-M18 correspond to a Linker 2 (ASTKG; SEQ ID NO:21; singleunderlined). Residues 249-276 of the first polypeptide chain of HIV-M18correspond to the heterodimer-promoting “E-coil” (EVAA

EK-EVAALEK-EVAALEK-EVAALEK; SEQ ID NO:31). Residues 277-279 of the firstpolypeptide chain of HIV-M18 correspond to a GGG Linker. Residues280-289 (underlined) of the first polypeptide chain of HIV-M18correspond to the Linker DKTHTCPPCP (SEQ ID NO:40; single underlined).Residues 290-506 of the first polypeptide chain of HIV-M18 correspond tothe IgG1 “knob-bearing” CH2-CH3 Domain (SEQ ID NO:48).

Since the VL Domain of CD3 mAb 1 M18 is the same as that of CD3 mAb 1,the amino acid sequence of the second polypeptide chain of HIV-M18 isthe same as that of the second polypeptide chain of the HIV-WT diabody(i.e., SEQ ID NO:186). Similarly, the amino acid sequence of the thirdpolypeptide chain of HIV-M18 is the same as that of the thirdpolypeptide chain of the CD123-WT diabody (i.e., SEQ ID NO:176).

14. Fourteenth Illustrative DART-B-Type Diabody CD19-WT (CD19×CD3 mAb 1)

An fourteenth illustrative DART-B-type diabody is similar to theabove-described HIV-WT diabody, but comprises CD19 mAb 1 in lieu of theA32 Binding Domain. This fourteenth illustrative DART-B-type diabody isdesignated “CD19-WT”.

Thus, the fourteenth illustrative DART-B-type diabody (CD19-WT) has afirst polypeptide chain that has the amino acid sequence (SEQ IDNO:191):

ENVLTQSPAT LSVTPGEKAT ITCRASQSVS YMHWYQQKPGQAPRLLIYDA SNRASGVPSR FSGSGSGTDH TLTISSLEAEDAATYYCFQG SVYPFTFGQG TKLEIK 

 

EVQLVE SGGGLVQPGG SLRLSCAASG FTFSTYAMNW VRQAPGKGLEWVGRIRSKYN NYATYYADSV K 

RFTISRDD SKNSLYLQMN SLKTEDTAVY YCVRHGNFGN SYVSWFAYWG QGTLVTVSS A STKGEVAACE KEVAALEKEV AALEKEVAAL EKGGG DKTHT CPPCPAPEAA GGPSVFLFPP KPKDILMISR TPEVTCVVVDVSHEDPEVKF NWYVDGVEVH NAKTKPREEQ YNSTYRVVSVLTVLHQDWLN GKEYKCKVSN KALPAPIEKT ISKAKGQPREPQVYTLPPSR EEMTKNQVSL WCLVKGFYPS DIAVEWESNGQPENNYKTTP PVLDSDGSFF LYSKLIVDKS RWQQGNVFSC SVMHEALHNH YTQKSLSLSP GK

Residues 1-106 of the first polypeptide chain of CD19-WT correspond tothe VL Domain of CD19 mAb 1 (SEQ ID NO:165). Residues 107-114 (doubleunderlined) of the first polypeptide chain of CD19-WT correspond toLinker 1 (GGGSGGGG; SEQ ID NO:16). Residues 115-239 of the firstpolypeptide chain of CD19-WT correspond to the VH Domain of CD3 mAb 1(SEQ ID NO:55), wherein Kabat position 65 (double underlined) is glycine(G). Residues 240-244 (underlined) of the first polypeptide chain ofCD19-WT correspond to a Linker 2 (ASTKG; SEQ ID NO:21; singleunderlined). Residues 245-272 of the first polypeptide chain of CD19-WTcorrespond to the heterodimer-promoting “E-coil” (EVAA

EK-EVAALEK-EVAALEK-EVAALEK; SEQ ID NO:31). Residues 273-275 of the firstpolypeptide chain of CD19-WT correspond to a GGG Linker (doubleunderlined). Residues 276-285 (single underlined) of the firstpolypeptide chain of CD19-WT correspond to the Linker DKTHTCPPCP (SEQ IDNO:40). Residues 286-502 of the first polypeptide chain of CD19-WTcorrespond to the IgG1 “knob-bearing” CH2-CH3 Domain (SEQ ID NO:48).

The second polypeptide chain of CD19-WT has the amino acid sequence (SEQID NO:192):

QAVVTQEPSL TVSPGGTVTL TCRSSTGAVT TSNYANWVQQKPGQAPRGLI GGTNKRAPWT PARFSGSLLG GKAALTITGAQAEDEADYYC ALWYSNLWVF GGGTKLTVLG GGGSGGGGQVTLRESGPALV KPTQTLTLTC TFSGFSLSTS GMGVGWIRQPPGKALEWLAH IWWDDDKRYN PALKSRLTIS KDTSKNQVFLTMTNMDPVDT ATYYCARMEL WSYYFDYWGQ GTTVTVSSASTKGKVAACKE KVAALKEKVA ALKEKVAALK E

Residues 1-110 of the second polypeptide chain of CD19-WT correspond tothe VL Domain of CD3 mAb 1 (SEQ ID NO:56). Residues 111-118 (doubleunderlined) of the second polypeptide chain of CD19-WT correspond toLinker 1 (GGGSGGGG; SEQ ID NO:16). Residues 119-238 of the secondpolypeptide chain of CD19-WT correspond to the VH Domain of CD19 mAb 1(SEQ ID NO:164). Residues 239-243 (underlined) of the second polypeptidechain of CD19-WT correspond to a Linker 2 (ASTKG; SEQ ID NO:21).Residues 244-271 of the second polypeptide chain of CD19-WT correspondto the heterodimer-promoting “K-coil” (KVAA

KE-KVAALKE-KVAALKE-KVAALKE; SEQ ID NO:32).

The third polypeptide chain of CD19-WT has the same amino acid sequenceas the third polypeptide chain of the CD123-WT diabody (i.e., SEQ IDNO:176).

15. Fifteenth Illustrative DART-B-Type Diabody CD19-M18 (CD19×CD3 mAb 1M18)

A fifteenth illustrative DART-B-type diabody is similar to theabove-described CD19-WT diabody, but contains the VH Domain of CD3 mAb 1M18. This fifteenth illustrative DART-B-type diabody is designated“CD19-M18”.

Thus, the fifteenth illustrative DART-B-type diabody (CD19-M18) has afirst polypeptide chain that has the amino acid sequence (SEQ IDNO:197):

ENVLIQSPAT LSVTPGEKAT ITCRASQSVS YMHWYQQKPG QAPRLLIYDA SNRASGVPSRFSGSGSGTDH TLTISSLEAE DAATYYCFQG SVYPFTFGQG TKLEIK 

 

EVQLVE SGGGLVQPGG SLRLSCAASG FTFSTYGMNW VRQAPGKGLE WVGRIRSKYN NYATYYADSVK 

RFTISRDD SKNSLYLQMN SLKTEDTAVY YCVRHGNFGN SYVSWFAYWG QGTLVTVSS A STKGEVAACE KEVAALEKEV AALEKEVAAL EKGGGDKTHT CPPCP APEAA GGPSVFLFPPKPKDTLMISR TPEVTCVVVD VSHEDPEVKF NWYVDGVEVE NAKTKPREEQ YNSTYRVVSVLTVLHQDWLN GKEYKCKVSN KALPAPIEKT ISKAKGQPRE PQVYTLPPSR EEMTKNQVSLWCLVKGFYPS DIAVEWESNG QPENNYKTTP PVLDSDGSFF LYSKLTVDKS RWQQGNVFSCSVMHEALHNH YTQKSLSLSP GK

Residues 1-106 of the first polypeptide chain of CD19-M18 correspond tothe VL Domain of CD19 mAb 1 (SEQ ID NO:165). Residues 107-114 (doubleunderlined) of the first polypeptide chain of CD19-M18 correspond toLinker 1 (GGGSGGGG; SEQ ID NO:16). Residues 115-239 of the firstpolypeptide chain of CD19-M18 correspond to the VH Domain of CD3 mAb 1M18 (SEQ ID NO:98), wherein Kabat position 65 (double underlined) isglycine (G). Residues 240-244 (underlined) of the first polypeptidechain of CD19-M18 correspond to a Linker 2 (ASTKG; SEQ ID NO:21; singleunderlined). Residues 245-272 of the first polypeptide chain of CD19-M18correspond to the heterodimer-promoting “E-coil” (EVAA

EK-EVAALEK-EVAALEK-EVAALEK; SEQ ID NO:31). Residues 273-275 of the firstpolypeptide chain of CD19-M18 correspond to a GGG Linker. Residues276-285 (single underlined) of the first polypeptide chain of CD19-M18correspond to the Linker DKTHTCPPCP (SEQ ID NO:40). Residues 286-502 ofthe first polypeptide chain of CD19-M18 correspond to the IgG1“knob-bearing” CH2-CH3 Domain (SEQ ID NO:48).

Since the VL Domain of CD3 mAb 1 M18 is the same as that of CD3 mAb 1,the amino acid sequence of the second polypeptide chain of CD19-M18 isthe same as that of the second polypeptide chain of the CD19-WT diabody(i.e., SEQ ID NO:192). Similarly, third polypeptide chain of CD19-M18has the same amino acid sequence as the third polypeptide chain of theCD123-WT diabody (i.e., SEQ ID NO:176).

16. Sixteenth Illustrative DART-B-Type Diabody CD19.1-M18 (CD19.1×CD3mAb 1 M18)

A sixteenth illustrative DART-B-type diabody is similar to theabove-described CD123-M18 diabody, but comprising a CD19 Binding Domain,containing the alternative VL Domain of CD19 mAb 1, in lieu of the CD123Binding Domain of CD123-M18. This illustrative DART-B-type diabody isdesignated “CD19.1-M18”. As indicated above, the VL Domain of CD3 mAb 1M18 has the same amino acid sequence as the VL Domain of CD3 mAb 1.

Thus, the sixteenth illustrative DART-B-type diabody (CD19.1-M18) has afirst polypeptide chain that has the amino acid sequence (SEQ IDNO:193):

ENVLTQSPAT LSVTPGEKVT ITCSASSSVS YMHWYQQKPGQAPRLLIYDT SKLASGVPSR FSGSGSGTDH FLTISSLEAEDAATYYCFQG SVYPFTFGQG TKLEIK 

 

EVQLVE SGGGLVQPGG SLRLSCAASG FTFSTYGMNW VRQAPGKGLEWVGRIRSKYN NYATYYADSV K 

RFTISRDD SKNSLYLQMN SLKTEDTAVY YCVRHGNFGN SYVSWFAYWG QGTLVTVSS G GCGGGEVAAL EKEVAALEKE VAALEKEVAA LEKGGG DKTH TCPPCPAPEA AGGPSVFLFP PKPKDTLMIS RTPEVTCVVVDVSHEDPEVK FNWYVDGVEV HNAKTKPREE QYNSTYRVVSVLTVLHQDWL NGKEYKCKVS NKALPAPIEK TISKAKGQPREPQVYTLPPS REEMTKNQVS LWCLVKGFYP SDIAVEWESNGQPENNYKTT PPVLDSDGSF FLYSKLTVDK SRWQQGNVFS CSVMHEALHN HYTQKSLSLS PGK

Residues 1-106 of the first polypeptide chain of CD19.1-M18 correspondto the alternative VL Domain of CD19 mAb 1 (SEQ ID NO:195). Residues107-114 (double underlined) of the first polypeptide chain of CD19.1-M18correspond to Linker 1 (GGGSGGGG; SEQ ID NO:16). Residues 115-239 of thefirst polypeptide chain of CD19.1-M18 correspond to the VH Domain of CD3mAb 1 M18 (SEQ ID NO:98), wherein Kabat position 65 (double underlined)is aspartate (D). Residues 240-245 (single underlined) of the firstpolypeptide chain of CD19.1-M18 correspond to a Linker 2 (GGCGGG; SEQ IDNO:17). Residues 246-273 of the first polypeptide chain of CD19.1-M18correspond to the heterodimer-promoting “E-coil”(EVAALEK-EVAALEK-EVAALEK-EVAALEK; SEQ ID NO:29). Residues 274-276 of thefirst polypeptide chain of CD19-M18 correspond to a GGG Linker. Residues277-286 (single underlined) of the first polypeptide chain of CD19.1-M18correspond to the Linker DKTHTCPPCP (SEQ ID NO:40). Residues 287-503 ofthe first polypeptide chain of CD19.1-M18 correspond to the IgG1“knob-bearing” CH2-CH3 Domain (SEQ ID NO:48).

The second polypeptide chain of CD19.1-M18 has the amino acid sequence(SEQ ID NO:194):

QAVVTQEPSL TVSPGGTVTL TCRSSTGAVT TSNYANWVQQKPGQAPRGLI GGTNKRAPWT PARFSGSLLG GKAALTITGAQAEDEADYYC ALWYSNLWVF GGGTKLTVLG 

QV TLRESGPALV KPTQTLTLTC TFSGFSLSTS GMGVGWIRQPPGKALEWLAH IWWDDDKRYN PALKSRLTIS KDTSKNQVFLTMTNMDPVDT ATYYCARMEL WSYYFDYWGQ GTTVTVSS GG CGGGKVAALK EKVAALKEKV AALKEKVAAL KE

Residues 1-110 of the second polypeptide chain of CD19.1-M18 correspondto the VL Domain of CD3 mAb 1 (SEQ ID NO:56). Residues 111-118 (doubleunderlined) of the second polypeptide chain of CD19.1-M18 correspond toLinker 1 (GGGSGGGG; SEQ ID NO:16). Residues 119-238 of the secondpolypeptide chain of CD19.1-M18 correspond to the VH Domain of CD19 mAb1 (SEQ ID NO:164). Residues 239-244 (single underlined) of the secondpolypeptide chain of CD19.1-M18 correspond to a Linker 2 (GGCGGG; SEQ IDNO:17). Residues 245-272 of the second polypeptide chain of CD19.1-M18correspond to the heterodimer-promoting “K-coil”(KVAALKE-KVAALKE-KVAALKE-KVAALKE; SEQ ID NO:30).

The amino acid sequence of the third polypeptide chain of CD19.1-M18 isthe same as that of the third polypeptide chain of the CD123-WT diabody(i.e., SEQ ID NO:176).

17. Seventeenth Illustrative DART-B-Type Diabody CD19.1-M13 (CD19.1×CD3mAb 1 M13)

A seventeenth illustrative DART-B-type diabody is similar to theabove-described CD19.1-M18 diabody, but contains the VH Domain of CD3mAb 1 M13 and is designated “CD19.1-M13”. As indicated above, the VLDomain of CD3 mAb 1 M13 has the same amino acid sequence as the VLDomain of CD3 mAb 1.

Thus, the seventeeth illustrative DART-B-type diabody (CD19.1-M13) has afirst polypeptide chain that has the amino acid sequence (SEQ IDNO:201):

ENVLTQSPAT LSVTPGEKVT ITCSASSSVS YMHWYQQKPGQAPRLLIYDT SKLASGVPSR FSGSGSGTDH FLTISSLEAEDAATYYCFQG SVYPFTFGQG TKLEIK 

  

EVQLVE SGGGLVQPGG SLRLSCAASG FTFSTYAMNW VRQAPGKGLEWVGRIRSKYN NYATYYADSV K 

RFTISRDD SKNSLYLQMN SLKTEDTAVY YCVRHGNFGN SYVSWFAEWG QGTLVTVSS G GCGGGEVAAL EKEVAALEKE VAALEKEVAA LEKGGG DKTH TCPPCPAPEA AGGPSVFLFP PKPKDTLMIS RTPEVTCVVVDVSHEDPEVK FNWYVDGVEV HNAKTKPREE QYNSTYRVVSVLTVLHQDWL NGKEYKCKVS NKALPAPIEK TISKAKGQPREPQVYTLPPS REEMTKNQVS LWCLVKGFYP SDIAVEWESNGQPENNYKTT PPVLDSDGSF FLYSKLTVDK SRWQQGNVFS CSVMHEALHN HYTQKSLSLS PGK

Residues 1-106 of the first polypeptide chain of CD19.1-M13 correspondto the alternative VL Domain of CD19 mAb 1 (SEQ ID NO:195). Residues107-114 (double underlined) of the first polypeptide chain of CD19.1-M13correspond to Linker 1 (GGGSGGGG; SEQ ID NO:16). Residues 115-239 of thefirst polypeptide chain of CD19.1-M18 correspond to the VH Domain of CD3mAb 1 M13 (SEQ ID NO:88), wherein Kabat position 65 (double underlined)is aspartate (D). Residues 240-245 (single underlined) of the firstpolypeptide chain of CD19.1-M13 correspond to a Linker 2 (GGCGGG; SEQ IDNO:17). Residues 246-273 of the first polypeptide chain of CD19.1-M13correspond to the heterodimer-promoting “E-coil”(EVAALEK-EVAALEK-EVAALEK-EVAALEK; SEQ ID NO:29). Residues 274-276 of thefirst polypeptide chain of CD19-M13 correspond to a GGG Linker. Residues277-286 (single underlined) of the first polypeptide chain of CD19.1-M13correspond to the Linker DKTHTCPPCP (SEQ ID NO:40). Residues 287-503 ofthe first polypeptide chain of CD19.1-M13 correspond to the IgG1“knob-bearing” CH2-CH3 Domain (SEQ ID NO:48).

Since the VL Domain of CD3 mAb 1 M13 is the same as that of CD3 mAb 1the amino acid sequence of the second polypeptide chain of CD19.1-M13 isthe same as that of the second polypeptide chain of the CD19.1-M18diabody (i.e., SEQ ID NO:194). The amino acid sequence of the thirdpolypeptide chain of CD19.1-M13 is the same as that of the thirdpolypeptide chain of the CD123-WT diabody (i.e., SEQ ID NO:176).

18. Eighteenth Illustrative DART-B-Type Diabody CD19.1-M17 (CD19.1×CD3mAb 1 M17)

An eighteenth illustrative DART-B-type diabody is similar to theabove-described CD19.1-M18 diabody, but contains the VH Domain of CD3mAb 1 M17 and is designated “CD19.1-M17”. As indicated above, the VLDomain of CD3 mAb 1 M17 has the same amino acid sequence as the VLDomain of CD3 mAb 1.

Thus, the eighteenth illustrative DART-B-type diabody (CD19.1-M17) has afirst polypeptide chain that has the amino acid sequence (SEQ IDNO:202):

ENVLTQSPAT LSVTPGEKVT ITCSASSSVS YMHWYQQKPGQAPRLLIYDT SKLASGVPSR FSGSGSGTDE FLTISSLEAEDAATYYCFQG SVYPFTFGQG TKLEIK 

 

EVQLVE SGGGLVQPGG SLRLSCAASG FTFSTTAMNW VRQAPGKGLEWVGRIRSKYN NYATYYADSV K 

RFTISRDD SKNSLYLQMN SLKTEDTAVY YCVRHGNFGN SYVSWFAYWG QGTLVTVSS G GCGGGEVAAL EKEVAALEKE VAALEKEVAA LEKGGG DKTH TCPPCPAPEA AGGPSVFLFP PKPKDTLMIS RTPEVTCVVVDVSHEDPEVK FNWYVDGVEV HNAKTKPREE QYNSTYRVVSVLTVLHQDWL NGKEYKCKVS NKALPAPIEK TISKAKGQPREPQVYTLPPS REEMTKNQVS LWCLVKGFYP SDIAVEWESNGQPENNYKTT PPVLDSDGSF FLYSKLTVDK SRWQQGNVFS CSVMHEALHN HYTQKSLSLS PGK

Residues 1-106 of the first polypeptide chain of CD19.1-M17 correspondto the alternative VL Domain of CD19 mAb 1 (SEQ ID NO:195). Residues107-114 (double underlined) of the first polypeptide chain of CD19.1-M17correspond to Linker 1 (GGGSGGGG; SEQ ID NO:16). Residues 115-239 of thefirst polypeptide chain of CD19.1-M17 correspond to the VH Domain of CD3mAb 1 M17 (SEQ ID NO:96), wherein Kabat position 65 (double underlined)is aspartate (D). Residues 240-245 (single underlined) of the firstpolypeptide chain of CD19.1-M17 correspond to a Linker 2 (GGCGGG; SEQ IDNO:17). Residues 246-273 of the first polypeptide chain of CD19.1-M17correspond to the heterodimer-promoting “E-coil”(EVAALEK-EVAALEK-EVAALEK-EVAALEK; SEQ ID NO:29). Residues 274-276 of thefirst polypeptide chain of CD19-M17 correspond to a GGG Linker. Residues277-286 (single underlined) of the first polypeptide chain of CD19.1-M17correspond to the Linker DKTHTCPPCP (SEQ ID NO:40). Residues 287-503 ofthe first polypeptide chain of CD19.1-M17 correspond to the IgG1“knob-bearing” CH2-CH3 Domain (SEQ ID NO:48).

Since the VL Domain of CD3 mAb 1 M17 is the same as that of CD3 mAb 1the amino acid sequence of the second polypeptide chain of CD19.1-M17 isthe same as that of the second polypeptide chain of the CD19.1-M18diabody (i.e., SEQ ID NO:194). The amino acid sequence of the thirdpolypeptide chain of CD19.1-M17 is the same as that of the thirdpolypeptide chain of the CD123-WT diabody (i.e., SEQ ID NO:176).

19. Nineteenth Illustrative DART-B-Type Diabody CD19.1-M19 (CD19.1×CD3mAb 1 M19)

A nineteenth illustrative DART-B-type diabody is similar to theabove-described CD19.1-M18 diabody, but contains the VH Domain of CD3mAb 1 M19 and is designated “CD19.1-M19”. As indicated above, the VLDomain of CD3 mAb 1 M19 has the same amino acid sequence as the VLDomain of CD3 mAb 1.

Thus, the nineteenth illustrative DART-B-type diabody (CD19.1-M19) has afirst polypeptide chain that has the amino acid sequence (SEQ IDNO:203):

ENVLTQSPAT LSVTPGEKVT ITCSASSSVS YMHWYQQKPGQAPRLLIYDT SKLASGVPSR FSGSGSGTDH FLTISSLEAEDAATYYCFQG SVYPFTFGQG TKLEIK 

 

EVQLVE SGGGLVQPGG SLRLSCAASG FTFSTYAMNW VRQAPGKGLEWVGRIRSKYN NYATYYADSV K 

RFTISRDD SKNSLYLQMN SLKTEDTAVY YCVRHKNIGN SYVSWFAYWG QGTLVTVSS G GCGGGEVAAL EKEVAALEKE VAALEKEVAA LEKGGG DKTH TCPPCPAPEA AGGPSVFLFP PKPKDTLMIS RTPEVTCVVVDVSHEDPEVK FNWYVDGVEV HNAKTKPREE QYNSTYRVVSVLTVLHQDWL NGKEYKCKVS NKALPAPIEK TISKAKGQPREPQVYTLPPS REEMTKNQVS LWCLVKGFYP SDIAVEWESNGQPENNYKTT PPVLDSDGSF FLYSKLTVDK SRWQQGNVFS CSVMHEALHN HYTQKSLSLS PGK

Residues 1-106 of the first polypeptide chain of CD19.1-M19 correspondto the alternative VL Domain of CD19 mAb 1 (SEQ ID NO:195). Residues107-114 (double underlined) of the first polypeptide chain of CD19.1-M19correspond to Linker 1 (GGGSGGGG; SEQ ID NO:16). Residues 115-239 of thefirst polypeptide chain of CD19.1-M19 correspond to the VH Domain of CD3mAb 1 M9 (SEQ ID NO:100), wherein Kabat position 65 (double underlined)is aspartate (D). Residues 240-245 (single underlined) of the firstpolypeptide chain of CD19.1-M19 correspond to a Linker 2 (GGCGGG; SEQ IDNO:17). Residues 246-273 of the first polypeptide chain of CD19.1-M19correspond to the heterodimer-promoting “E-coil”(EVAALEK-EVAALEK-EVAALEK-EVAALEK; SEQ ID NO:29). Residues 274-276 of thefirst polypeptide chain of CD19-M19 correspond to a GGG Linker. Residues277-286 (single underlined) of the first polypeptide chain of CD19.1-M19correspond to the Linker DKTHTCPPCP (SEQ ID NO:40). Residues 287-503 ofthe first polypeptide chain of CD19.1-M19 correspond to the IgG1“knob-bearing” CH2-CH3 Domain (SEQ ID NO:48).

Since the VL Domain of CD3 mAb 1 M19 is the same as that of CD3 mAb 1the amino acid sequence of the second polypeptide chain of CD19.1-M19 isthe same as that of the second polypeptide chain of the CD19.1-M18diabody (i.e., SEQ ID NO:194). The amino acid sequence of the thirdpolypeptide chain of CD19.1-M13 is the same as that of the thirdpolypeptide chain of the CD123-WT diabody (i.e., SEQ ID NO:176).

Additional CD19×CD3 DART-B-Type Diabodies specifically contemplated aresimilar to the above-described CD19-WT (see, the fourteenth illustrativeDART-B-Type Diabody) but will comprise the VH Domain of CD3 mAb 1 M13,M17, or M19. Such diabodies will comprise a first polypeptide chainhaving one of the following amino acid sequences:

SEQ ID NO:204 for such diabody comprising the VH Domain of CD3 mAb 1M13:

ENVLTQSPAT LSVTPGEKAT ITCRASQSVS YMHWYQQKPGQAPRLLIYDA SNRASGVPSR FSGSGSGTDH TLTISSLEAEDAATYYCFQG SVYPFTFGQG TKLEIK 

 

EVQLVE SGGGLVQPGG SLRLSCAASG FTFSTYAMNW VRQAPGKGLEWVGRIRSKYN NYATYYADSV K 

RFTISRDD SKNSLYLQMN SLKTEDTAVY YCVRHGNFGN SYVSWFAEWG QGTLVTVSS A STKGEVAACE KEVAALEKEV AALEKEVAAL EKGGG DKTHT CPPCPAPEAA GGPSVFLFPP KPKDTLMISR TPEVTCVVVDVSHEDPEVKF NWYVDGVEVH NAKTKPREEQ YNSTYRVVSVLTVLHQDWLN GKEYKCKVSN KALPAPIEKT ISKAKGQPREPQVYTLPPSR EEMTKNQVSL WCLVKGFYPS DIAVEWESNGQPENNYKTTP PVLDSDGSFF LYSKLTVDKS RWQQGNVFSC SVMHEALHNH YTQKSLSLSP GK

SEQ ID NO:205 for such diabody comprising the VH Domain of CD3 mAb 1M17:

ENVLTQSPAT LSVTPGEKAT ITCRASQSVS YMHWYQQKPGQAPRLLIYDA SNRASGVPSR FSGSGSGTDH TLTISSLEAEDAATYYCFQG SVYPFTFGQG TKLEIK 

 

EVQLVE SGGGLVQPGG SLRLSCAASG FTFSTTAMNW VRQAPGKGLEWVGRIRSKYN NYATYYADSV K 

RFTISRDD SKNSLYLQMN SLKTEDTAVY YCVRHGNFGN SYVSWFAYWG QGTLVTVSS A STKGEVAACE KEVAALEKEV AALEKEVAAL EKGGG DKTHT CPPCPAPEAA GGPSVFLFPP KPKDTLMISR TPEVTCVVVDVSHEDPEVKF NWYVDGVEVE NAKTKPREEQ YNSTYRVVSVLTVLHQDWLN GKEYKCKVSN KALPAPIEKT ISKAKGQPREPQVYTLPPSR EEMTKNQVSL WCLVKGFYPS DIAVEWESNGQPENNYKTTP PVLDSDGSFF LYSKLTVDKS RWQQGNVFSC SVMHEALENE YTQKSLSLSP GK

SEQ ID NO:206 for such diabody comprising the VH Domain of CD3 mAb 1M19:

ENVLTQSPAT LSVTPGEKAT ITCRASQSVS YMHWYQQKPGQAPRLLIYDA SNRASGVPSR FSGSGSGTDH TLTISSLEAEDAATYYCFQG SVYPFTFGQG TKLEIK 

 

EVQLVE SGGGLVQPGG SLRLSCAASG FTFSTYAMNW VRQAPGKGLEWVGRIRSKYN NYATYYADSV K 

RFTISRDD SKNSLYLQMN SLKTEDTAVY YCVRHKNIGN SYVSWFAYWG QGTLVTVSS A STKGEVAACE KEVAALEKEV AALEKEVAAL EKGGG DKTHT CPPCPAPEAA GGPSVFLFPP KPKDTLMISR TPEVTCVVVDVSHEDPEVKF NWYVDGVEVH NAKTKPREEQ YNSTYRVVSVLTVLHQDWLN GKEYKCKVSN KALPAPIEKT ISKAKGQPREPQVYTLPPSR EEMTKNQVSL WCLVKGFYPS DIAVEWESNGQPENNYKTTP PVLDSDGSFF LYSKLTVDKS RWQQGNVFSC SVMHEALHNH YTQKSLSLSP GK

The second polypeptide chains of such diabodies will have the same aminoacid sequence of CD19-WT (i.e., SEQ ID NO:192) and the third polypeptidechain of such diabodies will have the same amino acid sequence as thethird polypeptide chain of CD123-WT (i.e., SEQ ID NO:176).

As will be recognized in view of the instant disclosure, additionalDART-B-type diabodies having a binding site for an alternative DiseaseAntigens and/or having the CD3 Binding Domains of alternative variantanti-CD3 antibodies (i.e., vCD3-Binding Domains) may likewise beconstructed (by employing the VL and VH Domains of such antibodies).Similarly, as provided herein, alternative DART-B-type molecules maylikewise be constructed incorporating alternative Linkers and/oralternative Heterodimer-Promoting Domains.

Additional, exemplary molecules capable of mediating the redirectedkilling of a cell expressing a Disease Antigen (e.g., a tumor cell)which may be used in the methods of the present invention includebispecific molecules capable of binding: CD19 and CD3 (see, e.g., U.S.Pat. No. 7,235,641 and WO 2016/048938); CD123 and CD3 (see, e.g., Kuo,S. R. et al., (2012) “Engineering a CD123×CD3 Bispecific scFvImmunofusion For The Treatment Of Leukemia And Elimination Of LeukemiaStem Cells,” Protein Eng Des Sel. 25:561-9; WO 2015/026892; WO2016/086189); gpA33 and CD3 (e.g., WO 2015/026894); CEA and CD3 (e.g.,WO 2013/012414; WO 2017/118675); B7-H3 and CD3 (e.g., WO 2017/030926);HER2 and CD3 (e.g., WO 2012/143524); 5T4 and CD3 (e.g., WO 2015/184203and WO 2013/041687), and other molecules having a CD3 Binding Domain(see, e.g., etc., WO 2013/026835, WO 2013/158856, WO 2014/110601, WO2016/182751, WO 2017/053469). As will be recognized in view of theinstant disclosure, the vCD3-Binding Domains of the instant inventionmay be incorporated into such molecules.

C. TRIVALENT-Type Molecules

TRIVALENT-type molecules are trivalent molecules capable of binding upto three different epitopes. In particular, the TRIVALENT-type moleculesof the instant invention are capable of binding CD3 and a DiseaseAntigen (e.g., a Cancer or Infectious Disease Antigen) and may furtherbind an addition antigen such as an additional Disease Antigen (e.g., aCancer or Infectious Disease Antigen) or an additional antigen expressedon the surface of an effector cell (e.g., CD8), or may bind to a secondepitope of CD3 or a second epitope of the Disease Antigen.TRIVALENT-type molecules comprise an Fc Domain. Provided herein areillustrative TRIVALENT-type diabodies composed of four polypeptidechains and have one binding site for CD3, one binding site for theCancer Antigen CD123 or for the Cancer Antigen 5T4, and one binding sitefor CD8 (see, e.g., FIG. 6A). The illustrative TRIVALENT-type moleculesof the invention are generated using the first and second polypeptidechains of the DART-B-type diabodies provided above in combination withthe illustrative third and fourth polypeptide chains provided below,which provide the CD8 Binding Domain. The first and second polypeptidechains form the CD3 and DA Binding Domains while the third and fourthpolypeptide chains form the CD8 Binding Domain. The first and thirdpolypeptide chains form an Fc Domain.

The illustrative TRIVALENT-type molecules provide below each incorporatea third polypeptide chain having the amino acid sequence of SEQ IDNO:187:

QVQLVESGGG VVQPGRSLRL SCAASGFTFS DFGMNWVRQAPGKGLEWVAL IYYDGSNKFY ADSVKGRFTI SRDNSKNTLYLQMNSLRAED TAVYYCAKPH YDGYYHFFDS WGQGTLVTVSSASTKGPSVF PLAPSSKSTS GGTAALGCLV KDYFPEPVTVSWNSGALTSG VHTFPAVLQS SGLYSLSSVV TVPSSSLGTQTYICNVNHKP SNTKVDKRVE PKSCDKTHTC PPCPAPEAAGGPSVFLFPPK PKDTLMISRT PEVTCVVVDV SHEDPEVKFNWYVDGVEVHN AKTKPREEQY NSTYRVVSVL TVLHQDWLNGKEYKCKVSNK ALPAPIEKTI SKAKGQPREP QVYTLPPSREEMTKNQVSLS CAVKGFYPSD IAVEWESNGQ PENNYKTTPPVLDSDGSFFL VSKLTVDKSR WQQGNVFSCS VMHEALHNRY TQKSLSLSPG K

Residues 1-121 of the third polypeptide chain of such illustrativeTRIVALENT-type molecules correspond to the VH Domain of the anti-CD8antibody TRX2 (SEQ ID NO:120). Residues 121-219 of the third polypeptidechain of such illustrative TRIVALENT-type molecule correspond to an IgG1CH1 Domain (SEQ ID NO:1). Residues 220-234 of the third polypeptidechain of such illustrative TRIVALENT-type molecule correspond to an IgG1Hinge Domain (SEQ ID NO:5). Residues 235-451 correspond to the IgG1“hole-bearing” CH2-CH3 Domain (SEQ ID NO:50).

The illustrative TRIVALENT-type molecules described below eachincorporate a fourth polypeptide chain having the amino acid sequence ofSEQ ID NO:188:

DIQMTQSPSS LSASVGDRVT ITCKGSQDIN NYLAWYQQKPGKAPKLLIYN TDILHTGVPS RFSGSGSGTD FTFTISSLQPEDIATYYCYQ YNNGYTFGQG TKVEIKRTVA APSVFIFPPSDEQLKSGTAS VVCLLNNFYP REAKVQWKVD NALQSGNSQESVTEQDSKDS TYSLSSTLTL SKADYEKEKV YACEVTEQGL SSPVTKSFNR GEC

Residues 1-106 of the fourth polypeptide chain of such illustrativeTRIVALENT-type molecules correspond to the VL Domain of the anti-CD8antibody TRX2 (SEQ ID NO:121). Residues 107-213 correspond to a CL KappaDomain (SEQ ID NO:14).

The SEQ ID NOs. of the polypeptide chains of Illustrative TRIVALENT-typemolecules are summarized in Table 10.

TABLE 10 CD123 × CD3 × CD8 TRIDENT Molecules Disease Polypeptide ChainTRIDENT- Antigen- CD3 First Second Third Fourth Type Binding BindingCD123/CD3 Binding CD8 Binding No. Domain Domain Domains DomainsDesignation 1 CD123 CD3 mAb 1 SEQ ID SEQ ID SEQ ID SEQ ID T-CD123-WT mAb1 NO: 174 NO: 175 NO: 187 NO: 188 2 CD3 mAb 1 SEQ ID T-CD123-M1 M1 NO:177 3 CD3 mAb 1 SEQ ID T-CD123-M2 M2 NO: 178 4 CD3 mAb 1 SEQ IDT-CD123-M18 M18 NO: 179

1. First Illustrative TRIVALENT-Type Molecule T-CD123-WT (CD123 mAb1×CD3 mAb 1×TRX2)

A first illustrative TRIVALENT-type molecule (designated “T-CD123-WT”)contains the VH and VL Domains of CD123 mAb 1, the VH and VL Domains ofCD3 mAb 1, and the VH and VL Domains of the anti-CD8 antibody TRX2. Asindicated above, the amino acid sequence of the first polypeptide chainis the same as that of the above-described CD123-WT diabody (SEQ IDNO:174). Similarly, the amino acid sequence of the second polypeptidechain is the same as that of the above-described CD123-WT diabody (SEQID NO:175). Also indicated above, the amino acid sequences of the thirdand fourth polypeptide chains of all the illustrative TRIVALENT-typemolecules are SEQ ID NO:187 and SEQ ID NO:188, respectively.

2. Second Illustrative TRIVALENT-Type Molecule T-CD123-M1 (CD123 mAb1×CD3 mAb 1 M1×TRX2)

A second illustrative TRIVALENT-type molecule (designated “T-CD123-M1”)contains the VH and VL Domains of CD123 mAb 1, the VH and VL Domains ofCD3 mAb 1 M1, and the VH and VL Domains of TRX2. As indicated above, theamino acid sequence of the first polypeptide chain is the same as thatof the above-described CD123-M1 diabody (SEQ ID NO:177). Similarly, theamino acid sequence of the second polypeptide chain is the same as thatof the above-described CD123-WT diabody (SEQ ID NO:175). Also asindicated above, the amino acid sequences of the third and fourthpolypeptide chains of all the illustrative TRIVALENT-type molecules areSEQ ID NO:187 and SEQ ID NO:188, respectively.

3. Third Illustrative TRIVALENT-Type Molecule T-CD123-M2 (CD123 mAb1×CD3 mAb 1 M2×TRX2)

A third illustrative TRIVALENT-type molecule designated T-CD123-M2 bindscontains the VH and VL Domains of CD123 mAb 1, the VH and VL Domains ofCD3 mAb 1 M2, and the VH and VL Domains of TRX2. As indicated above, theamino acid sequence of the first polypeptide chain is the same as thatof the above-described CD123-M2 diabody (SEQ ID NO:178). Similarly, theamino acid sequence of the second polypeptide chain is the same as thatof the above-described CD123-WT diabody (SEQ ID NO:175). Also indicatedabove, the amino acid sequences of the third and fourth polypeptidechains of all the illustrative TRIVALENT-type molecules are SEQ IDNO:187 and SEQ ID NO:188 respectively.

4. Fourth Illustrative TRIVALENT-Type Molecule T-CD123-M18 (CD123 mAb1×CD3 mAb 1 M18×TRX2)

A fourth illustrative TRIVALENT-type molecule designated T-CD123-M18binds contains the VH and VL Domains of CD123 mAb 1, the VH and VLDomains of CD3 mAb 1 M18, and the VH and VL Domains of TRX2. Asindicated above, the amino acid sequence of the first polypeptide chainis the same as that of the above-described CD123-M18 diabody (SEQ IDNO:179). Similarly, the amino acid sequence of the second polypeptidechain is the same as that of the above-described CD123-WT diabody (SEQID NO:175). Also indicated above, the amino acid sequences of the thirdand fourth polypeptide chains of all the illustrative TRIVALENT-typemolecules are SEQ ID NO:187 and SEQ ID NO:188, respectively.

As will be recognized in view of the instant disclosure, additionalTRIVALENT-type diabodies having a binding site for an alternativeDisease Antigens and/or having the CD3 Binding Domains of alternativevariant anti-CD3 antibodies (i.e., vCD3-Binding Domains) may likewise beconstructed (by employing the VL and VH Domains of such antibodies).Similarly, as provided herein, alternative TRIVALENT-type molecules maylikewise be constructed incorporating alternative Linkers and/oralternative Heterodimer-Promoting Domains.

Additional, exemplary molecules capable of mediating the redirectedkilling of a cell expressing a Disease Antigen (e.g., a tumor cell)which may be used in the methods of the present invention includetrivalent molecules capable of binding: B7-H3, CD3 and CD8 (see, e.g.,WO 2015/184203); 5T4, CD3 and CD8 (see, e.g., WO 2015/184203); ROR1, CD3and CD8 (see, e.g., WO 2015/184203 and WO 2017/106061); HIV, CD3 and CD8(see, e.g., WO 2015/184203; WO2017/011413; and WO2017/011414); gpA33,CD3 and DR5 (see, e.g., WO 2015/184207); EphA2, CD3 and DR5 (see, e.g.,WO 2015/184207); gpA33, CD3 and EphA2 (see, e.g., WO 2015/184207); andother trivalent molecules (see, e.g., WO 2016/105450; WO 2016/115274; WO2017/180913). As will be recognized in view of the instant disclosure,the vCD3-Binding Domains of the instant invention may be incorporatedinto such molecules.

VIII. Methods of Production

The molecules of the present invention are most preferably producedthrough the recombinant expression of nucleic acid molecules that encodesuch polypeptides, as is well-known in the art.

Polypeptides of the invention may be conveniently prepared usingsolid-phase peptide synthesis (Merrifield, B. (1986) “Solid PhaseSynthesis,” Science 232(4748):341-347; Houghten, R. A. (1985) “GeneralMethod For The Rapid Solid-Phase Synthesis Of Large Numbers Of Peptides:Specificity Of Antigen-Antibody Interaction At The Level Of IndividualAmino Acids,” Proc. Natl. Acad. Sci. (U.S.A.) 82(15):5131-5135; Ganesan,A. (2006) “Solid-Phase Synthesis In The Twenty-First Century,” Mini Rev.Med. Chem. 6(1):3-10).

Antibodies may be made recombinantly and expressed using any methodknown in the art. Antibodies may be made recombinantly by firstisolating the antibodies made from host animals, obtaining the genesequence, and using the gene sequence to express the antibodyrecombinantly in host cells (e.g., CHO cells). Another method that maybe employed is to express the antibody sequence in plants (e.g.,tobacco) or transgenic milk. Suitable methods for expressing antibodiesrecombinantly in plants or milk have been disclosed (see, for example,Peeters et al. (2001) “Production Of Antibodies And Antibody FragmentsIn Plants,” Vaccine 19:2756; Lonberg, N. et al. (1995) “Human AntibodiesFrom Transgenic Mice,” Int. Rev. Immunol 13:65-93; and Pollock et al.(1999) “Transgenic Milk As A Method For The Production Of RecombinantAntibodies,” J. Immunol. Methods 231:147-157). Suitable methods formaking derivatives of antibodies, e.g., humanized, single-chain, etc.are known in the art, and have been described above. In anotheralternative, antibodies may be made recombinantly by phage displaytechnology (see, for example, U.S. Pat. Nos. 5,565,332; 5,580,717;5,733,743; 6,265,150; and Winter, G. et al. (1994) “Making Antibodies ByPhage Display Technology,” Annu. Rev. Immunol. 12.433-455).

Vectors containing polynucleotides of interest (e.g., polynucleotidesencoding the polypeptide chains of the Binding Molecules of the presentinvention) can be introduced into the host cell by any of a number ofappropriate means, including electroporation, transfection employingcalcium chloride, rubidium chloride, calcium phosphate, DEAE-dextran, orother substances; microprojectile bombardment; lipofection; andinfection (e.g., where the vector is an infectious agent such asvaccinia virus). The choice of introducing vectors or polynucleotideswill often depend on features of the host cell.

Any host cell capable of overexpressing heterologous DNAs can be usedfor the purpose of expressing a polypeptide or protein of interest.Non-limiting examples of suitable mammalian host cells include but arenot limited to COS, HeLa, and CHO cells.

The invention includes polypeptides comprising an amino acid sequence ofa binding molecule of this invention. The polypeptides of this inventioncan be made by procedures known in the art. The polypeptides can beproduced by proteolytic or other degradation of the antibodies, byrecombinant methods (i.e., single or fusion polypeptides) as describedabove or by chemical synthesis. Polypeptides of the antibodies,especially shorter polypeptides up to about 50 amino acids, areconveniently made by chemical synthesis. Methods of chemical synthesisare known in the art and are commercially available.

The invention includes variants of the disclosed Binding Molecules,including functionally equivalent polypeptides that do not significantlyaffect the properties of such molecules as well as variants that haveenhanced or decreased activity. Modification of polypeptides is routinepractice in the art and need not be described in detail herein. Examplesof modified polypeptides include polypeptides with conservativesubstitutions of amino acid residues, one or more deletions or additionsof amino acids which do not significantly deleteriously change thefunctional activity, or use of chemical analogs. Amino acid residuesthat can be conservatively substituted for one another include but arenot limited to: glycine/alanine; serine/threonine;valine/isoleucine/leucine; asparagine/glutamine; aspartic acid/glutamicacid; lysine/arginine; and phenylalanine/tyrosine. These polypeptidesalso include glycosylated and non-glycosylated polypeptides, as well aspolypeptides with other posttranslational modifications, such as, forexample, glycosylation with different sugars, acetylation, andphosphorylation. Preferably, the amino acid substitutions would beconservative, i.e., the substituted amino acid would possess similarchemical properties as that of the original amino acid. Suchconservative substitutions are known in the art, and examples have beenprovided above. Amino acid modifications can range from changing ormodifying one or more amino acids to complete redesign of a region, suchas the Variable Domain. Changes in the Variable Domain can alter bindingaffinity and/or specificity. Other methods of modification include usingcoupling techniques known in the art, including, but not limited to,enzymatic means, oxidative substitution and chelation. Modifications canbe used, for example, for attachment of labels for immunoassay, such asthe attachment of radioactive moieties for radioimmunoassay. Modifiedpolypeptides are made using established procedures in the art and can bescreened using standard assays known in the art.

In one embodiment, a fusion polypeptide is provided that comprises aLight Chain, a Heavy Chain or both a Light and Heavy Chain. In anotherembodiment, the fusion polypeptide contains a heterologousimmunoglobulin constant region. In another embodiment, the fusionpolypeptide contains a VH and a VL Domain of an antibody produced from apublicly-deposited hybridoma. For purposes of this invention, anantibody fusion protein contains one or more polypeptide domains thatspecifically bind CD3, or to both CD3 and to a Disease Antigen, andwhich contains another amino acid sequence to which it is not attachedin the native molecule, for example, a heterologous sequence or ahomologous sequence from another region.

The present invention particularly encompasses such Binding Molecules(e.g., antibodies, diabodies, trivalent Binding Molecules, etc.)conjugated to a diagnostic or therapeutic moiety. For diagnosticpurposes, the Binding Molecules of the invention may be coupled to adetectable substance. Such Binding Molecules are useful for monitoringand/or prognosing the development or progression of a disease as part ofa clinical testing procedure, such as determining the efficacy of aparticular therapy. Examples of detectable substances include variousenzymes (e.g., horseradish peroxidase, beta-galactosidase, etc.),prosthetic groups (e.g., avidin/biotin), fluorescent materials (e.g.,umbelliferone, fluorescein, or phycoerythrin), luminescent materials(e.g., luminol), bioluminescent materials (e.g., luciferase oraequorin), radioactive materials (e.g., carbon-14, manganese-54,strontium-85 or zinc-65), positron emitting metals, and nonradioactiveparamagnetic metal ions. The detectable substance may be coupled orconjugated either directly to the binding molecule or indirectly,through an intermediate (e.g., a Linker) using techniques known in theart.

For therapeutic purposes, the Binding Molecules of the invention may beconjugated to a therapeutic moiety such as a cytotoxin, (e.g., acytostatic or cytocidal agent), a therapeutic agent or a radioactivemetal ion, e.g., alpha-emitters. A cytotoxin or cytotoxic agent includesany agent that is detrimental to cells such as, for example, Pseudomonasexotoxin, Diptheria toxin, a botulinum toxin A through F, ricin abrin,saporin, and cytotoxic fragments of such agents. A therapeutic agentincludes any agent having a therapeutic effect to prophylactically ortherapeutically treat a disorder. Such therapeutic agents may be may bechemical therapeutic agents, protein or polypeptide therapeutic agents,and include therapeutic agents that possess a desired biologicalactivity and/or modify a given biological response. Examples oftherapeutic agents include alkylating agents, angiogenesis inhibitors,anti-mitotic agents, hormone therapy agents, and antibodies useful forthe treatment of cell proliferative disorders. The therapeutic moietymay be coupled or conjugated either directly to the binding molecule orindirectly, through an intermediate (e.g., a Linker) using techniquesknown in the art.

IX. Uses of the Binding Molecules of the Present Invention

As discussed above, molecules capable of binding CD3 and a DiseaseAntigen are capable of mediating the redirected cell killing of a targetcell (i.e., a cancer cell, or a pathogen-infected cell) that expressessuch Disease Antigen on its cell surface. Such molecules may be used fortherapeutic purposes, for example in subjects with cancer or aninfection. Thus, Binding Molecules of the present invention have theability to treat any disease or condition associated with orcharacterized by the expression of a Disease Antigen, particularly aCancer Antigen or a Pathogen-Associated Antigen, on the surface of suchtarget cell. Thus, without limitation, the Binding Molecules of thepresent invention may be employed in the treatment of cancer,particularly a cancer characterized by the expression of a CancerAntigen. The Binding Molecules of the present invention may be employedin the treatment of infection, particularly an infection characterizedby the expression of a Pathogen-Associated Antigen.

In particular, the present invention encompasses such methods whereinthe molecule capable of binding CD3 comprises an Epitope-Binding Domainof an antibody that is capable of binding CD3 and also comprises anEpitope-Binding Domain capable of binding a Disease Antigen (inparticular a Cancer Antigen or a Pathogen-Associated Antigen) on thesurface of a target cell so as to mediate the redirected killing of thetarget cell (for example, by mediating redirected cell killing (e.g.,redirected T-cell cytotoxicity)).

In a specific embodiment, the molecule capable of binding CD3 and theDisease Antigen is a bispecific antibody, or a molecule comprising theEpitope-Binding Domains thereof, (including a bispecific scFv a BiTe, aTandAb).

In a specific embodiment, the molecule capable of binding CD3 and theDisease Antigen is a bispecific diabody.

In a specific embodiment, the molecule capable of binding CD3 and theDisease Antigen is a trivalent binding molecule.

“Providing a therapy” or “treating” refers to any administration of acomposition that is associated with any indicia of beneficial or desiredresult, including, without limitation, any clinical result such asdecreasing symptoms resulting from the disease, attenuating a symptom ofinfection (e.g., viral load, fever, pain, sepsis, etc.) a shrinking ofthe size of a tumor (in the cancer context, for example, a tumor ofbreast, gastric or prostate cancer), a retardation of cancer cellgrowth, a delaying of the onset, development or progression ofmetastasis, a decreasing of a symptom resulting from the disease, anincreasing of the quality of life of the recipient subject, a decreasingof the dose of other medications being provided to treat a subject'sdisease, an enhancing of the effect of another medication such as viatargeting and/or internalization, a delaying of the progression of thedisease, and/or a prolonging of the survival of recipient subject.

Subjects for treatment include animals, most preferably mammalianspecies such as non-primate (e.g., bovine, equine, feline, canine,rodent, etc.) or a primate (e.g., monkey such as, a cynomolgus monkey,human, etc.). In a preferred embodiment, the subject is a human.

Exemplary disorders that may be treated by various embodiments of thepresent invention include, but are not limited to, proliferativedisorders, cell proliferative disorders, and cancer (especially a cancerexpressing a Cancer Antigen bound by a molecule capable of mediatingredirected cell killing), pathogen-associated diseases (especially achronic viral infection associated with expression of aPathogen-Associated Antigen bound by a molecule capable of mediatingredirected cell killing). In various embodiments, the inventionencompasses methods and compositions for treatment, prevention ormanagement of a disease or disorder in a subject, comprisingadministering to the subject a therapeutically effective amount theBinding Molecules of the present invention. Such molecules areparticularly useful for the prevention, inhibition, reduction of growth,or regression of primary tumors, and metastasis of tumors, and forreducing pathogen load, or eliminating pathogen-infected cells. Althoughnot intending to be bound by a particular mechanism of action, suchmolecules may mediate effector function against target cells, promotethe activation of the immune system against target cells, cross-linkcell surface antigens and/or receptors on target cells and enhanceapoptosis or negative growth regulatory signaling, or a combinationthereof, resulting in clearance and/or reduction in the number of targetcells.

The cancers that may be treated by molecules of the present invention,and by the methods of the present invention, include, but are notlimited to: adrenal cancer, bladder cancer, breast cancer, colorectalcancer, gastric cancer, glioblastoma, kidney cancer, non-small-cell lungcancer, hematological cancer, multiple myeloma, melanoma, ovariancancer, pancreatic cancer, prostate cancer, skin cancer, renal cellcarcinoma, testicular cancer, and uterine cancer.

In particular, the CD19×CD3 Binding Molecules, CD19×CD3×CD8 BindingMolecules, CD123×CD3 Binding Molecules and CD123×CD3×CD8 BindingMolecules of the present invention may be used in the treatment of ahematological cancer including but not limited to: acute myeloidleukemia (AML), chronic myelogenous leukemia (CML), myelodysplasticsyndrome (MDS), acute B lymphoblastic leukemia (B-ALL), chroniclymphocytic leukemia (CLL), including Richter's syndrome or Richter'stransformation of CLL, hairy cell leukemia (HCL), blastic plasmacytoiddendritic cell neoplasm (BPDCN), non-Hodgkin's lymphoma (NHL), includingmantle cell lymphoma (MCL) and small lymphocytic lymphoma (SLL),Hodgkin's lymphoma, systemic mastocytosis, and Burkitt's lymphoma.

Pathogen-associated diseases that may be treated by the LAG-3-BindingMolecules of the present invention include chronic viral, bacterial,fungal and parasitic infections. Chronic infections that may be treatedby the LAG-3-Binding Molecules of the present invention includeEpstein-Barr Virus, Hepatitis A Virus (HAV); Hepatitis B Virus (HBV);Hepatitis C Virus (HCV); herpes viruses (e.g. HSV-1, HSV-2, HHV-6, CMV),Human Immunodeficiency Virus (HIV), Vesicular Stomatitis Virus (VSV),Bacilli, Citrobacter, Cholera, Diphtheria, Enterobacter, Gonococci,Helicobacter pylori, Klebsiella, Legionella, Meningococci, mycobacteria,Pseudomonas, Pneumonococci, rickettsia bacteria, Salmonella, Serratia,Staphylococci, Streptococci, Tetanus, Aspergillus (fumigatus, niger,etc.), Blastomyces dermatitidis, Candida (albicans, krusei, glabrata,tropicalis, etc.), Cryptococcus neoformans, Genus Mucorales (mucor,absidia, rhizopus), Sporothrix schenkii, Paracoccidioides brasiliensis,Coccidioides immitis, Histoplasma capsulatum, Leptospirosis, Borreliaburgdorferi, helminth parasite (hookworm, tapeworms, flukes, flatworms(e.g. Schistosomia), Giardia lambia, trichinella, Dientamoeba Fragilis,Trypanosoma brucei, Trypanosoma cruzi, and Leishmania donovani).

X. Pharmaceutical Compositions

The present invention encompasses compositions comprising a moleculecapable of binding CD3 and also capable of binding to a Disease Antigen(e.g., a DA×CD3 Binding Molecule, including, for example, a DA×CD3×CD8Binding Molecule, a DA×CD3×DA Binding Molecule, etc.). The compositionsof the invention include bulk drug compositions useful in themanufacture of pharmaceutical compositions (e.g., impure or non-sterilecompositions) and pharmaceutical compositions (i.e., compositions thatare suitable for administration to a subject or patient) that can beused in the preparation of unit dosage forms. Such compositions comprisea prophylactically or therapeutically effective amount of a moleculecapable of binding CD3 and also capable of binding to a Disease Antigenso as to be capable of mediating the redirected killing of a target cell(e.g., a cancer cell, a pathogen-infected cell, etc.), or a combinationof such agents and a pharmaceutically acceptable carrier. Preferably,compositions of the invention comprise a prophylactically ortherapeutically effective amount of the Binding Molecules of the presentinvention and a pharmaceutically acceptable carrier. In a preferredaspect, such compositions are substantially purified (i.e.,substantially free from substances that limit its effect or produceundesired side effects).

Various formulations of such compositions may be used foradministration. In addition to the pharmacologically active agent(s),the compositions of the present invention may contain suitablepharmaceutically acceptable carriers comprising excipients andauxiliaries that are well-known in the art and are relatively inertsubstances that facilitate administration of a pharmacologicallyeffective substance or which facilitate processing of the activecompounds into preparations that can be used pharmaceutically fordelivery to the site of action. For example, an excipient can give formor consistency, or act as a diluent. Suitable excipients include but arenot limited to stabilizing agents, wetting and emulsifying agents, saltsfor varying osmolarity, encapsulating agents, buffers, and skinpenetration enhancers.

In a specific embodiment, the term “pharmaceutically acceptable” meansapproved by a regulatory agency of the Federal or a state government orlisted in the U.S. Pharmacopeia or other generally recognizedpharmacopeia for use in animals, and more particularly in humans. Theterm “carrier” refers to a diluent, adjuvant (e.g., Freund's adjuvant(complete and incomplete), excipient, or vehicle with which thetherapeutic is administered. Generally, the ingredients of compositionsof the invention are supplied either separately or mixed together inunit dosage form, for example, as a dry lyophilized powder or water freeconcentrate in a hermetically sealed container such as an ampoule orsachette indicating the quantity of active agent. Where the compositionis to be administered by infusion, it can be dispensed with an infusionbottle containing sterile pharmaceutical grade water or saline. Wherethe composition is administered by injection, an ampoule of sterilewater for injection or saline can be provided so that the ingredientsmay be mixed prior to administration.

The invention also provides a pharmaceutical pack or kit comprising oneor more containers filled with a binding molecule of the presentinvention, alone or with such pharmaceutically acceptable carrier.Additionally, one or more other prophylactic or therapeutic agentsuseful for the treatment of a disease can also be included in thepharmaceutical pack or kit. The invention also provides a pharmaceuticalpack or kit comprising one or more containers filled with one or more ofthe ingredients of the pharmaceutical compositions of the invention.Optionally associated with such container(s) can be a notice in the formprescribed by a governmental agency regulating the manufacture, use orsale of pharmaceuticals or biological products, which notice reflectsapproval by the agency of manufacture, use or sale for humanadministration.

The present invention provides kits that can be used in the abovemethods. A kit can comprise any of the Binding Molecules of the presentinvention. The kit can further comprise one or more other prophylacticand/or therapeutic agents useful for the treatment of cancer, in one ormore containers.

XI. Methods of Administration

The compositions of the present invention may be provided for thetreatment, prophylaxis, and amelioration of one or more symptomsassociated with a disease, disorder or infection by administering to asubject an effective amount of the pharmaceutical compositions of thepresent invention. In a preferred aspect, such compositions aresubstantially purified (i.e., substantially free from substances thatlimit its effect or produce undesired side effects). In a specificembodiment, the subject is an animal, preferably a mammal such asnon-primate (e.g., bovine, equine, feline, canine, rodent, etc.) or aprimate (e.g., monkey such as, a cynomolgus monkey, human, etc.). In apreferred embodiment, the subject is a human.

Methods of administering a molecule or composition of the inventioninclude, but are not limited to, parenteral administration (e.g.,intradermal, intramuscular, intraperitoneal, intravenous andsubcutaneous), epidural, and mucosal (e.g., intranasal and oral routes).In a specific embodiment, the Binding Molecules of the present inventionare administered intramuscularly, intravenously, or subcutaneously. Thecompositions may be administered by any convenient route, for example,by infusion or bolus injection, by absorption through epithelial ormucocutaneous linings (e.g., oral mucosa, rectal and intestinal mucosa,etc.) and may be administered together with other biologically activeagents. Administration can be systemic or local.

The invention also provides that preparations of the Binding Moleculesof the present invention are packaged in a hermetically sealed containersuch as an ampoule or sachette indicating the quantity of the molecule.In one embodiment, such molecules are supplied as a dry sterilizedlyophilized powder or water free concentrate in a hermetically sealedcontainer and can be reconstituted, e.g., with water or saline to theappropriate concentration for administration to a subject. Preferably,the Binding Molecules of the present invention are supplied as a drysterile lyophilized powder in a hermetically sealed container.

The lyophilized preparations of the Binding Molecules of the presentinvention should be stored at between 2° C. and 8° C. in their originalcontainer and the molecules should be administered within 12 hours,preferably within 6 hours, within 5 hours, within 3 hours, or within 1hour after being reconstituted. In an alternative embodiment, suchmolecules are supplied in liquid form in a hermetically sealed containerindicating the quantity and concentration of the molecule, fusionprotein, or conjugated molecule. Preferably, such Binding Molecules,when provided in liquid form, are supplied in a hermetically sealedcontainer.

The amount of such preparations of the invention that will be effectivein the treatment, prevention or amelioration of one or more symptomsassociated with a disorder can be determined by standard clinicaltechniques. The precise dose to be employed in the formulation will alsodepend on the route of administration, and the seriousness of thecondition, and should be decided according to the judgment of thepractitioner and each patient's circumstances. Effective doses may beextrapolated from dose-response curves derived from in vitro or animalmodel test systems.

As used herein, an “effective amount” of a pharmaceutical composition isan amount sufficient to effect beneficial or desired results including,without limitation, clinical results such as decreasing symptomsresulting from the disease, attenuating a symptom of infection (e.g.,viral load, fever, pain, sepsis, etc.) or a symptom of cancer (e.g., theproliferation, of cancer cells, tumor presence, tumor metastases, etc.),thereby increasing the quality of life of those suffering from thedisease, decreasing the dose of other medications required to treat thedisease, enhancing the effect of another medication such as viatargeting and/or internalization, delaying the progression of thedisease, and/or prolonging survival of individuals. When applied to anindividual active ingredient, administered alone, the term refers tothat ingredient alone. When applied to a combination, the term refers tocombined amounts of the active ingredients that result in thetherapeutic effect, whether administered in combination, serially, orsimultaneously.

An effective amount can be administered in one or more administrations.For purposes of this invention, an effective amount of drug, compound,or pharmaceutical composition is an amount sufficient: to kill and/orreduce the proliferation of cancer cells, and/or to eliminate, reduceand/or delay the development of metastasis from a primary site ofcancer; or to reduce the proliferation of (or the effect of) aninfectious pathogen and to reduce and/or delay the development of thepathogen-mediated disease, either directly or indirectly. In someembodiments, an effective amount of a drug, compound, or pharmaceuticalcomposition may or may not be achieved in conjunction with another drug,compound, or pharmaceutical composition. Thus, an “effective amount” maybe considered in the context of administering one or morechemotherapeutic agents, and a single agent may be considered to begiven in an effective amount if, in conjunction with one or more otheragents, a desirable result may be or is achieved. While individual needsvary, determination of optimal ranges of effective amounts of eachcomponent is within the skill of the art.

For the Binding Molecules encompassed by the invention, the dosageadministered to a patient is preferably determined based upon the bodyweight (kg) of the recipient subject. For the Binding Moleculesencompassed by the invention, the dosage administered to a patient istypically from about 0.01 μg/kg to about 30 mg/kg or more of thesubject's body weight.

The dosage and frequency of administration of a binding molecule of thepresent invention may be reduced or altered by enhancing uptake andtissue penetration of the molecule by modifications such as, forexample, lipidation.

The dosage of a binding molecule of the invention administered to apatient may be calculated for use as a single agent therapy.Alternatively, the molecule may be used in combination with othertherapeutic compositions and the dosage administered to a patient arelower than when said molecules are used as a single agent therapy.

The pharmaceutical compositions of the invention may be administeredlocally to the area in need of treatment; this may be achieved by, forexample, and not by way of limitation, local infusion, by injection, orby means of an implant, said implant being of a porous, non-porous, orgelatinous material, including membranes, such as sialastic membranes,or fibers. Preferably, when administering a molecule of the invention,care must be taken to use materials to which the molecule does notabsorb.

The compositions of the invention can be delivered in a vesicle, inparticular a liposome (See Langer (1990) “New Methods Of Drug Delivery,”Science 249:1527-1533); Treat et al., in LIPOSOMES IN THE THERAPY OFINFECTIOUS DISEASE AND CANCER, Lopez-Berestein and Fidler (eds.), Liss,New York, pp. 353-365 (1989); Lopez-Berestein, ibid., pp. 3 17-327).

Treatment of a subject with a therapeutically or prophylacticallyeffective amount of a binding molecule of the present invention caninclude a single treatment or, preferably, can include a series oftreatments. In a preferred example, a subject is treated with apharmaceutical composition of the invention for between about 1 to 10weeks, preferably between 2 to 8 weeks, more preferably between about 3to 7 weeks, and even more preferably for about 4, 5, or 6 weeks. Thepharmaceutical compositions of the invention can be administered once aday with such administration occurring once a week, twice a week, onceevery two weeks, once a month, once every six weeks, once every twomonths, twice a year or once per year, etc. Alternatively, thepharmaceutical compositions of the invention can be administered twice aday with such administration occurring once a week, twice a week, onceevery two weeks, once a month, once every six weeks, once every twomonths, twice a year or once per year, etc. Alternatively, thepharmaceutical compositions of the invention can be administered threetimes a day with such administration occurring once a week, twice aweek, once every two weeks, once a month, once every six weeks, onceevery two months, twice a year or once per year, etc. It will also beappreciated that the effective dosage of the molecules used fortreatment may increase or decrease over the course of a particulartreatment.

XII. Specific Embodiments of the Invention

Specific embodiments of the of the invention include Embodiments E1-E27:

-   -   E1. A DA×CD3 Binding Molecule comprising a CD3-Binding Domain        capable of binding an epitope of CD3 and a Disease        Antigen-Binding Domain capable of binding an epitope of a        Disease Antigen, wherein said CD3-Binding Domain comprises:        -   (I) (A) a CDR_(H)1 Domain comprising an amino acid sequence            selected from the group consisting of SEQ ID NO:99, SEQ ID            NO:91, SEQ ID NO:93, SEQ ID NO:95 and SEQ ID NO:97;            -   (B) a CDR_(H)2 Domain comprising the amino acid sequence                of SEQ ID NO:58;            -   (C) a CDR_(H)3 Domain comprising the amino acid sequence                of SEQ ID NO:59;            -   (D) a CDR_(L)1 Domain comprising the amino acid sequence                of SEQ ID NO:60;            -   (E) a CDR_(L)2 Domain comprising the amino acid sequence                of SEQ ID NO:61; and            -   (F) a CDR_(L)3 Domain comprising the amino acid sequence                of SEQ ID NO:62; or        -   (II) (A) a CDR_(H)1 Domain comprising the amino acid            sequence of SEQ ID NO:57;            -   (B) a CDR_(H)2 Domain comprising the amino acid sequence                of SEQ ID NO:58;            -   (C) a CDR_(H)3 Domain comprising an amino acid sequence                selected from the group consisting of SEQ ID NO:69, SEQ                ID NO:71, SEQ ID NO:73, SEQ ID NO:75, SEQ ID NO:77, SEQ                ID NO:79, SEQ ID NO:81, SEQ ID NO:83, SEQ ID NO:85, SEQ                ID NO:87, SEQ ID NO:89, SEQ ID NO:101, SEQ ID NO:103,                SEQ ID NO:105 and SEQ ID NO:107;            -   (D) a CDR_(L)1 Domain comprising the amino acid sequence                of SEQ ID NO:60;            -   (E) a CDR_(L)2 Domain comprising the amino acid sequence                of SEQ ID NO:61; and            -   (F) a CDR_(L)3 Domain comprising the amino acid sequence                of SEQ ID NO:62; or        -   (III) (A) a CDR_(H)1 Domain comprising the amino acid            sequence of SEQ ID NO:57;            -   (B) a CDR_(H)2 Domain comprising the amino acid sequence                of SEQ ID NO:58;            -   (C) a CDR_(H)3 Domain comprising the amino acid sequence                of SEQ ID NO:59;            -   (D) a CDR_(L)1 Domain comprising the amino acid sequence                of SEQ ID NO:60;            -   (E) a CDR_(L)2 Domain comprising the amino acid sequence                of SEQ ID NO:61; and            -   (F) a CDR_(L)3 Domain comprising an amino acid sequence                selected from the group consisting of SEQ ID NO:109 or                SEQ ID NO:111; or        -   (IV) (A) a CDR_(H)1 Domain comprising the amino acid            sequence of SEQ ID NO:57;            -   (B) a CDR_(H)2 Domain comprising the amino acid sequence                of SEQ ID NO:58;            -   (C) a CDR_(H)3 Domain comprising the amino acid sequence                of SEQ ID NO:59;            -   (D) a CDR_(L)1 Domain comprising the amino acid sequence                of SEQ ID NO:60;            -   (E) a CDR_(L)2 Domain comprising an amino acid sequence                selected from the group consisting of SEQ ID NO:113 and                SEQ ID NO:115; and            -   (F) a CDR_(L)3 Domain comprising the amino acid sequence                of SEQ ID NO:62.    -   E2. The DA×CD3 Binding Molecule of E1, wherein said CD3-Binding        Domain comprises:        -   (I) (A) a VL Domain comprising the amino acid sequence of            SEQ ID NO:56;            -   (B) a VH Domain comprising an amino acid sequence                selected from the group consisting of SEQ ID NO:98, SEQ                ID NO:68, SEQ ID NO:70, SEQ ID NO:72, SEQ ID NO:74, SEQ                ID NO:76, SEQ ID NO:78, SEQ ID NO:80, SEQ ID NO:82, SEQ                ID NO:84, SEQ ID NO:86, SEQ ID NO:88, SEQ ID NO:90, SEQ                ID NO: 92, SEQ ID NO:94, SEQ ID NO:96, SEQ ID NO:100,                SEQ ID NO:102, SEQ ID NO:104 and SEQ ID NO:106; or        -   (II) (A) a VL Domain comprising an amino acid sequence            selected from the group consisting of SEQ ID NO:108, SEQ ID            NO:110, SEQ ID NO:112; and SEQ ID NO:114;            -   (B) a VH Domain comprising an amino acid sequence of SEQ                ID NO:55.    -   E3. The DA×CD3 Binding Molecule of E1 or E2, wherein said DA×CD3        Binding Molecule is a bispecific antibody, a bispecific diabody,        a bispecific scFv, a bispecific TandAb, or a trivalent binding        molecule.    -   E4. The DA×CD3 Binding Molecule of any one of E1-E3, wherein        said DA×CD3 Binding Molecule is capable of binding more than one        Disease Antigen and/or a different cell surface molecule of an        effector cell.    -   E5. The DA×CD3 Binding Molecule of any one of E1-E4, wherein        said Disease Antigen is a Cancer Antigen.    -   E6. The DA×CD3 Binding Molecule of any one of E1-E4, wherein        said Disease Antigen is a Pathogen-Associated Antigen.    -   E7. The DA×CD3 Binding Molecule, of any one of E4-E6, wherein        said different cell surface molecule of an effector cell is CD2,        CD8, CD16, TCR, NKp46, or NKG2D.    -   E8. The DA×CD3 Binding Molecule of E5 or E7, wherein said Cancer        Antigen is selected from the group consisting of the Cancer        Antigens: 19.9, 4.2, ADAM-9, AH6, ALCAM, B1, B7-H3, BAGE,        beta-catenin, blood group ALe^(b)/Le^(y), Burkitt's lymphoma        antigen-38.13, C14, CA125, Carboxypeptidase M, CD5, CD19, CD20,        CD22, CD23, CD25, CD27, CD28, CD33, CD36, CD40/CD154, CD45,        CD56, CD46, CD52, CD56, CD79a/CD79b, CD103, CD123, CD317, CDK4,        CEA, CEACAM5/CEACAM6, C017-1A, CO-43, CO-514, CTA-1, CTLA-4,        Cytokeratin 8, D1.1, D₁56-22, DR5, E₁ series, EGFR, an Ephrin        receptor, EphA2, Erb, GAGE, a GD2/GD3/GM2 ganglioside, GICA        19-9, gp100, Gp37, gp75, gpA33, HER2/neu, HMFG, Human        Papillomavirus-E6/Human Papillomavirus-E7, HMW-MAA, I antigen,        IL13Rα2, Integrin β6, JAM-3, KID3, KID31, KS 1/4 pan-carcinoma        antigen, L6, L20, LEA, LUCA-2, M1:22:25:8, M18, M39, MAGE, MART,        mesothelin, MUC-1, MUM-1, Myl, N-acetylglucosaminyltransferase,        neoglycoprotein, NS-10, OFA-1, OFA-2, Oncostatin M, p15, p97,        PEM, PEMA, PIPA, PSA, PSMA, prostatic acid phosphate, R₂₄, ROR1,        a sphingolipid, SSEA-1, SSEA-3, SSEA-4, sTn, the T-cell receptor        derived peptide, T₅A₇, TAG-72, TL5, TNF-receptor, TNF-7        receptor, TRA-1-85, a Transferrin Receptor, 5T4, TSTA, VEGF, a        VEGF Receptor, VEP8, VEP9, VIM-D5, and Y hapten, Le^(y).    -   E9. The DA×CD3 Binding Molecule of E8, wherein said Disease        Antigen is B7-H3, CEACAM5/CEACAM6, EGRF, EphA2, gpA33, HER2/neu,        VEGF, 5T4, IL13Rα2, CD123, CD19, or ROR1.    -   E10. The DA×CD3 Binding Molecule of E6 or E7, wherein said        Pathogen-Associated Antigen is selected from the group        consisting of the Pathogen-Associated Antigens: Herpes Simplex        Virus infected cell protein (ICP)47, Herpes Simplex Virus gD,        Epstein-Barr Virus LMP-1, Epstein-Barr Virus LMP-2A,        Epstein-Barr Virus LMP-2B, Human Immunodeficiency Virus gp160,        Human Immunodeficiency Virus gp120, Human Immunodeficiency Virus        gp41, Human Papillomavirus E6, Human Papillomavirus E7, human        T-cell leukemia virus gp64, human T-cell leukemia virus gp46,        and human T-cell leukemia virus gp21.    -   E11. The DA×CD3 Binding Molecule of any one of E1-E10, wherein        said DA×CD3 Binding Molecule comprises: a first polypeptide        chain and a second polypeptide chain, covalently bonded to one        another, wherein:        -   (A) the first polypeptide chain comprises, in the N-terminal            to C-terminal direction:            -   (i) a Domain 1, comprising:                -   (1) a sub-Domain (1A), which comprises a VL Domain                    of a monoclonal antibody capable of binding to said                    epitope of a Disease Antigen (VL_(DA)); and                -   (2) a sub-Domain (1B), which comprises a VH Domain                    of a monoclonal antibody capable of binding to said                    epitope of CD3 (VH_(CD3));                -   wherein said sub-Domains 1A and 1B are separated                    from one another by a peptide Linker; and            -   (ii) a Domain 2, wherein said Domain 2 is a                Heterodimer-Promoting Domain;        -   (B) the second polypeptide chain comprises, in the            N-terminal to C-terminal direction:            -   (i) a Domain 1, comprising:                -   (1) a sub-Domain (1A), which comprises a VL Domain                    of said monoclonal antibody capable of binding to                    said epitope of CD3 (VL_(CD3)); and                -   (2) a sub-Domain (1B), which comprises a VH Domain                    of said monoclonal antibody capable of binding to                    said epitope of a Disease Antigen (VH_(DA));                -   wherein said sub-Domains 1A and 1B are separated                    from one another by a peptide Linker;            -   (ii) a Domain 2, wherein said Domain 2 is a                Heterodimer-Promoting Domain, wherein said                Heterodimer-Promoting Domain of said first and said                second polypeptide chains are different;        -   and wherein:        -   (a) the VL Domain of the first polypeptide chain and the VH            Domain of the second polypeptide chain associate to form the            Disease Antigen-Binding Domain, and the VH Domain of the            first polypeptide chain and the VL Domain of the second            polypeptide chain associate to form the CD3-Binding Domain;            or        -   (b) the VL Domain of the first polypeptide chain and the VH            Domain of the second polypeptide chain associate to form the            CD3-Binding Domain, and the VH Domain of the first            polypeptide chain and the VL Domain of the second            polypeptide chain associate to form the Disease            Antigen-Binding Domain.    -   E12. The DA×CD3 Binding Molecule of E11, wherein:        -   (a) said Heterodimer-Promoting Domain of said first            polypeptide chain is an E-coil Domain, and said            Heterodimer-Promoting Domain of said second polypeptide            chain is a K-coil Domain; or        -   (b) said Heterodimer-Promoting Domain of said first            polypeptide chain is a K-coil Domain, and said            Heterodimer-Promoting Domain of said second polypeptide            chain is an E-coil Domain.    -   E13. The DA×CD3 Binding Molecule of E11 or E12, wherein the        first or second polypeptide chain additionally comprises a        Domain 3 comprising a CH2 and CH3 Domain of an immunoglobulin Fc        Domain.    -   E14. The DA×CD3 Binding Molecule of E13, wherein said DA×CD3        Binding Molecule further comprises a third polypeptide chain        comprising a CH2 and CH3 Domain of an immunoglobulin Fc Domain.    -   E15. The DA×CD3 Binding Molecule of any one of E11-E14, wherein        said DA×CD3 Binding Molecule further comprises a CD8-Binding        Domain.    -   E16. The DA×CD3 Binding Molecule of any one of E11-E15, wherein        said DA×CD3 Binding Molecule comprises:        -   (I) (A) a first polypeptide comprising SEQ ID NO:179;            -   (B) a second polypeptide comprising SEQ ID NO:175; and            -   (C) a third polypeptide comprising SEQ ID NO:176; or        -   (II) (A) a first polypeptide comprising SEQ ID NO:184;            -   (B) a second polypeptide comprising SEQ ID NO:181; and            -   (C) a third polypeptide comprising SEQ ID NO:176; or        -   (III) (A) a first polypeptide comprising SEQ ID NO:196;            -   (B) a second polypeptide comprising SEQ ID NO:186; and            -   (C) a third polypeptide comprising SEQ ID NO:176; or        -   (IV) (A) a first polypeptide comprising SEQ ID NO:197;            -   (B) a second polypeptide comprising SEQ ID NO:192; and            -   (C) a third polypeptide comprising SEQ ID NO:176; or        -   (V) (A) a first polypeptide comprising SEQ ID NO:193;            -   (B) a second polypeptide comprising SEQ ID NO:194; and            -   (C) a third polypeptide comprising SEQ ID NO:176; or        -   (VI) (A) a first polypeptide comprising SEQ ID NO:179;            -   (B) a second polypeptide comprising SEQ ID NO:175;            -   (C) a third polypeptide comprising SEQ ID NO:187; and            -   (D) a fourth polypeptide comprising SEQ ID NO:188; or        -   (VII) (A) a first polypeptide comprising SEQ ID NO:184;            -   (B) a second polypeptide comprising SEQ ID NO:181;            -   (C) a third polypeptide comprising SEQ ID NO:187; and            -   (D) a fourth polypeptide comprising SEQ ID NO:188; or        -   (VIII) (A) a first polypeptide comprising SEQ ID NO:196;            -   (B) a second polypeptide comprising SEQ ID NO:186;            -   (C) a third polypeptide comprising SEQ ID NO:187; and            -   (D) a fourth polypeptide comprising SEQ ID NO:188; or        -   (IX) (A) a first polypeptide comprising SEQ ID NO:193;            -   (B) a second polypeptide comprising SEQ ID NO:194;            -   (C) a third polypeptide comprising SEQ ID NO:187; and            -   (D) a fourth polypeptide comprising SEQ ID NO:188.    -   E17. A pharmaceutical composition that comprises the DA×CD3        Binding Molecule of any of E1-E16 and a pharmaceutically        acceptable carrier.    -   E18. A method for the treatment of a disease, comprising        administering to a subject in need thereof a therapeutically        effective amount of the DA×CD3 Binding Molecule of any of E1-E16        or the pharmaceutical composition of E17.    -   E19. The method of E18, wherein said disease is cancer.    -   E20. The method of E19, wherein said cancer is selected from the        group consisting of adrenal cancer, bladder cancer, breast        cancer, colorectal cancer, gastric cancer, glioblastoma, kidney        cancer, non-small-cell lung cancer, hematological cancer,        multiple myeloma, melanoma, ovarian cancer, pancreatic cancer,        prostate cancer, skin cancer, renal cell carcinoma, testicular        cancer, and uterine cancer.    -   E21. The method of E18, wherein said disease is a        pathogen-associated disease.    -   E22. The method of E21, wherein said Pathogen-Associated Antigen        is selected from the group consisting of the Pathogen-Associated        Antigens: Herpes Simplex Virus infected cell protein (ICP)47,        Herpes Simplex Virus gD, Epstein-Barr Virus LMP-1, Epstein-Barr        Virus LMP-2A, Epstein-Barr Virus LMP-2B, Human Immunodeficiency        Virus gp160, Human Immunodeficiency Virus gp120, Human        Immunodeficiency Virus gp41, Human Papillomavirus E6, Human        Papillomavirus E7, human T-cell leukemia virus gp64, human        T-cell leukemia virus gp46, and human T-cell leukemia virus        gp21.    -   E23. The DA×CD3 Binding Molecule of any of E1-E16 or the        pharmaceutical composition of E16 for use in the treatment of a        disease.    -   E24. The DA×CD3 Binding Molecule or pharmaceutical composition        of E23, wherein said disease is cancer.    -   E25. The DA×CD3 Binding Molecule or pharmaceutical composition        of E24, wherein said cancer is selected from the group        consisting of adrenal cancer, bladder cancer, breast cancer,        colorectal cancer, gastric cancer, glioblastoma, kidney cancer,        non-small-cell lung cancer, hematological cancer, multiple        myeloma, melanoma, ovarian cancer, pancreatic cancer, prostate        cancer, skin cancer, renal cell carcinoma, testicular cancer,        and uterine cancer.    -   E26. The DA×CD3 Binding Molecule or pharmaceutical composition        of E23, wherein said disease is a pathogen-associated disease.    -   E27. The DA×CD3 Binding Molecule or pharmaceutical composition        of E26, wherein said Pathogen-Associated Antigen is selected        from the group consisting of the Pathogen-Associated Antigens:        Herpes Simplex Virus infected cell protein (ICP)47, Herpes        Simplex Virus gD, Epstein-Barr Virus LMP-1, Epstein-Barr Virus        LMP-2A, Epstein-Barr Virus LMP-2B, Human Immunodeficiency Virus        gp160, Human Immunodeficiency Virus gp120, Human        Immunodeficiency Virus gp41, Human Papillomavirus E6, Human        Papillomavirus E7, human T-cell leukemia virus gp64, human        T-cell leukemia virus gp46, and human T-cell leukemia virus        gp21.

EXAMPLES

Having now generally described the invention, the same will be morereadily understood through reference to the following examples, whichare provided by way of illustration and are not intended to be limitingof the present invention unless specified.

Example 1 Evaluation of CD3 mAb 1 M3-CD3 mAb 1 M26

A CD3 mAb 1 scFv saturation-mutant library at 29 CDR positions wasconstructed and expressed in E. coli (XL-1 Blue). A multi-well formatwas used to produce soluble scFv. scFv-containing supernatants werecaptured on anti-His surface and screened for binding to recombinant CD3(ε/γ chain Fos/Jun heterodimer) using an Attana biosensor to identifyvCD3-Binding Domains.

Variants of the CD123×CD3 DART-A-type diabody (designated DART-A-WT;amino acid sequences provided above) were generated comprising the VHand VL Domains of the identified scFvs. Thus, a panel of DART-A-typediabodies, designated DART-A-M1-DART-A-M26, comprising vCD3-BindingDomains (such vCD3-Binding Domains being respectively designated “CD3mAb 1 M1-CD3 mAb 1 M26”) were generated. The CD3-binding kinetics ofDART-A-M1-DART-A-M26 were measured by BIACORE® and compared toDART-A-WT. Table 11 summarizes the CD3-binding kinetics of DART-A-WT andthe DART-A-type diabodies comprising the vCD3-Binding Domains of CD3 mAb1 M1-CD3 mAb 1 M26, ranked by k_(a) (R denotes k_(a), k_(d), or k_(D)ratio of variant DART-A-type diabody (comprising a vCD3-Binding Domain)to DART-A-WT (comprising the rCD3-Binding Domain of CD3 mAb 1)).

TABLE 11 CD3-Binding Kinetics of CD123 × CD3 DART- A-Type Diabodies CD3mAb 1 M1 -- CD3 mAb 1 M26 CD3 mAb1 Substitution ka kd KD Variant(Relative to CD3 Mab-1) (M⁻¹s⁻¹) R (s⁻¹) R (M) R M25 G50D/VL 1.54 × 10⁵0.3 1.92 × 10⁻² 4.7 1.25 × 10⁻⁷ 15 M18 A33G/VH 2.87 × 10⁵ 0.6 5.88 ×10⁻² 14 2.05 × 10⁻⁷ 25 M14 T31D/VH 3.08 × 10⁵ 0.6 1.43 × 10⁻² 3.5 4.66 ×10⁻⁸ 5.7 M26 K53G/VL 3.19 × 10⁵ 0.6 1.74 × 10⁻² 4.3 5.44 × 10⁻⁸ 6.7 M13Y102E/VH 3.21 × 10⁵ 0.6 2.20 × 10⁻² 5.4 6.86 × 10⁻⁸ 8.4 M16 Y32D/VH 3.47× 10⁵ 0.7 1.04 × 10⁻¹ 25 3.01 × 10⁻⁷ 37 M15 T31E/VH 3.52 × 10⁵ 0.7 3.75× 10⁻² 9.2 1.07 × 10⁻⁷ 13 M1 S100dT/VH 4.08 × 10⁵ 0.8 6.50 × 10⁻² 161.59 × 10⁻⁷ 19 M3 G99I/VH 4.49 × 10⁵ 0.9 8.30 × 10⁻³ 2 1.85 × 10⁻⁸ 2.3M23 L95E/VL 4.59 × 10⁵ 0.9 1.24 × 10⁻² 3 2.69 × 10⁻⁸ 3.3 CD3 Mab-1Wild-Type 5.00 × 10⁵ 1 4.09 × 10⁻³ 1 8.17 × 10⁻⁹ 1 M24 L95Q/VL 5.94 ×10⁵ 1.2 8.61 × 10⁻³ 2.1 1.45 × 10⁻⁸ 1.8 M6 Y100bQ/VH 6.99 × 10⁵ 1.4 2.01× 10⁻² 4.9 2.88 × 10⁻⁸ 3.5 M10 F98I/VH 7.17 × 10⁵ 1.4 3.10 × 10⁻² 7.64.33 × 10⁻⁸ 5.3 M19 G96K/F98I/VH 7.53 × 10⁵ 1.5 4.11 × 10⁻² 10 5.46 ×10⁻⁸ 6.7 M4 Y100bA/VH 7.90 × 10⁵ 1.6 2.03 × 10⁻² 5 2.57 × 10⁻⁸ 3.1 M17Y32T/VH 8.37 × 10⁵ 1.7 4.78 × 10⁻² 12 5.72 × 10⁻⁸ 7 M12 W100eY/VH 8.66 ×10⁵ 1.7 1.11 × 10⁻² 2.7 1.28 × 10⁻⁸ 1.6 M7 G96D/VH 1.02 × 10⁶ 2 2.29 ×10⁻² 5.6 2.25 × 10⁻⁸ 2.8 M8 G96E/VH 1.13 × 10⁶ 2.3 7.84 × 10⁻³ 1.9 6.91× 10⁻⁹ 0.8 M5 Y100bG/VH 1.31 × 10⁶ 2.6 2.80 × 10⁻² 6.8 2.14 × 10⁻⁸ 2.6M11 W100eF/VH 1.39 × 10⁶ 2.8 2.67 × 10⁻² 6.5 1.92 × 10⁻⁸ 2.4 M9 G96K/VH2.15 × 10⁶ 4.3 7.16 × 10⁻³ 1.8 3.33 × 10⁻⁹ 0.4 M22 G96K/W100eY/VH 2.37 ×10⁶ 4.7 1.56 × 10⁻² 3.8 6.60 × 10⁻⁹ 0.8 M21 G96K/W100eF/VH 2.45 × 10⁶4.9 1.03 × 10⁻² 2.5 4.18 × 10⁻⁹ 0.5 M2 G96K/S100dT/VH 3.07 × 10⁶ 6.13.91 × 10⁻² 9.6 1.27 × 10⁻⁸ 1.6 M20 G96K/Y100bG/VH 3.87 × 10⁶ 7.7 7.32 ×10⁻² 18 1.89 × 10⁻⁸ 2.3

The ability of DART-A-M1-DART-A-M26 (comprising vCD3-Binding Domains) tomediate T-cell redirected cell killing was measured in CTL assay andcompared to DART-A-WT (comprising the rCD3-Binding Domain). Briefly, theDART-A-type diabodies were incubated with effector Pan-T-cells andMOLM-13 target tumor cells, at an effector:target cell ratio of 5:1 for18 and 42 hours and the EC₅₀ was determined by measuring the release oflactate dehydrogenase (LDH) into the media by damaged cells (e.g., byusing the CytoTox 96® Non-Radioactive Cytotoxicity Assay Kit (Promega)that quantitatively measures LDH release, or similar). A 4420×CD3fluorescein-binding DART-A-type diabody having the CD3 Binding Domain ofCD3 mAb 1 was employed as a negative control. In addition, the stabilityof the DART-A-type diabodies was evaluated by measuring the Tm usingDSF. Representative cytotoxicity curves for DART-A-WT; DART-A-M1;DART-A-M2; DART-A-M15; DART-A-M17; DART-A-M18; DART-A-M19; andDART-A-M20 are presented in FIG. 7A. Table 12 summarizes thecytotoxicity (i.e., T-cell redirected killing activity) ofDART-A-M1-DART-A-M26, ranked by 18-hour EC₅₀ (R denotes ratio of variantDART-A-type diabody (comprising a vCD3-Binding domain) to DART-A-WT(comprising the rCD3-Binding Domain of CD3 mAb 1); ΔTm denotes change inTm as compared to WT (Tm=63° C.). The relationship of the kineticparameters and cytolytic potency is plotted in FIGS. 7B-7D (FIG. 7B:affinity vs cytolysis (18 hour EC50), FIG. 7C: association rate vscytolysis (EC50). FIG. 7C: dissociation rate vs cytolysis (EC50)). TheCD3 mAb1 (∘), M18 (▪), M2 (▴) and M1 (▾) variants are indicated.

TABLE 12 Cytotoxicity Of Antibodies CD3 mAb 1 M1 - CD3 mAb 1 M26 MOLM-13(E:T = 5:1) CD3 EC₅₀ max % EC₅₀ max % R mAb 1 Substitution (ng/mL) RCytoTox (ng/mL) R CytoTox ΔTm Variant (Relative to CD3 Mab-1) 18 Hour 42Hour (° C.) M21 G96K/W100eF/VH 0.026 0.3 36.54 0 0 29.92 −1 M8 G96E/VH0.069 0.9 38.43 0.005 1.7 29.35 1 M22 G96K/W100eY/VH 0.072 0.9 38.260.01 3.5 31.58 −0.5 M9 G96K/VH 0.075 1 38.25 0.001 0.4 30.46 0 CD3 Mab-1Wild-Type 0.079 1 38.97 0.003 1 29.13 — M3 G99I/VH 0.108 1.4 39.69 0.0062.3 28.58 0 M6 Y100bQ/VH 0.162 2.1 41.86 0.011 3.9 29.46 1 M4 Y100bA/VH0.176 2.2 41.69 0.011 3.9 29.8 1 M11 W100eF/VH 0.223 2.8 48.55 0.016 5.832.43 0.5 M5 Y100bG/VH 0.225 2.9 40.75 0.011 3.8 28.41 1 M20G96K/Y100bG/VH 0.259 3.3 37.48 0.038 13.5 30.89 −0.5 M7 G96D/VH 0.3043.8 41.38 0.03 10.8 32.54 1 M24 L95Q/VL 0.34 4.3 49.41 0.031 11.1 31.120.5 M12 W100eY/VH 0.372 4.7 49.72 0.067 24 31.54 0.5 M23 L95E/VL 0.4215.3 49.29 0.061 21.6 31.37 −0.5 M10 F98I/VH 0.426 5.4 39.26 0.095 33.729.87 0 M26 K53G/VL 0.444 5.6 44.23 0.078 27.9 28.19 0 M14 T31D/VH 0.4926.2 44.69 0.11 39.3 31.36 1.5 M25 G50D/VL 0.585 7.4 46.56 0.1 35.7 30.410 M19 G96K/F98I/VH 0.68 8.6 41.01 0.11 39.1 32.83 −0.5 M13 Y102E/VH1.223 15.5 45.08 0.203 72.3 31.48 −0.5 M17 Y32T/VH 1.283 16.2 44.230.126 44.9 30.69 −0.5 M15 T31E/VH 4.164 52.7 42.74 0.975 347.2 32.05 1.5M18 A33G/VH 4.687 59.3 41.3 0.91 324 30.58 0.5 M2 G96K/S100dT/VH 8.113102.7 32.33 0.693 246.9 32.77 −1 M1 S100dT/VH 19.64 248.6 32.32 2.336831.9 32.37 0 M16 Y32D/VH NA NA NA NA NA NA −0.5

As indicated in Tables 11-12, the DART-A-M1-DART-A-M26 variantsdisplayed a range of binding kinetics and CTL activity, while retainingtheir thermal stability. Such DA×CD3 Binding Molecules, and theirvCD3-Binding Domains, are useful for modulating CD3 binding, redirectedT-cell killing activity, and/or T-cell stimulation activity.

Example 2 CTL Activity and Cytokine Release of Representative Variants

The cytotoxic (CTL) activity and cytokine release profile of arepresentative set of DART-A-type diabodies comprising variant CD3 mAb 1VL or VH Domains was assessed by incubating DART-A-WT; DART-A-M2;DART-A-M7; DART-A-M13; and DART-A-M15 in the presence of Pan-T-cellseffector cells and MV-4-11 leukemia target tumor cells at aneffector:target cell ratio of 5:1 24 hours. The percentage cytotoxicity(i.e., cell killing) and/or EC₅₀ was determined by measuring the releaseof lactate dehydrogenase (LDH) into the media by damaged cells (e.g., byusing the CytoTox 96® Non-Radioactive Cytotoxicity Assay Kit (Promega)that quantitatively measures LDH release, or similar) and is plotted inFIG. 8A. Cytokines released into the supernatant during the CTL wasmeasured (e.g., using Enzyme-Linked ImmunoSpot (ELISPOT) assay ormilliplex cytokine assay). Cytokine release is plotted in FIGS. 8B-8E(FIG. 8B: INF-γ, FIG. 8C: TNF-α, FIG. 8D: IL-6, and FIG. 8E: IL-2). TheEC₅₀ Values for cytotoxicity and cytokine release are provided in Table13. A 4420×CD3 fluorescein-binding DART-A-type diabody having the CD3Binding Domain of CD3 mAb 1 was employed as a negative control(NegCtrl).

TABLE 13 DART-A- EC50- Type EC₅₀CTL EC₅₀INF-γ EC₅₀TNFα EC₅₀IL-6 IL2 WT0.029 0.11 0.76 0.13 0.85 M2 0.92 1.7 4.7 10 27 M7 0.052 0.98 6.6 1.17.0 M13 0.12 0.83 0.12 1.3 7.5 M15 0.39 3.5 18 7.5 30

The results from these studies show that DA×CD3 Binding Moleculescomprising vCD3-Binding Domains displaying having altered affinityretain cytolytic activity and exhibit one or more reduced cytokineresponses (max response and/or EC50) as compared to a DA×CD3 BindingMolecule comprising the rCD3-Binding Domain.

Example 3 Generation of DART-B-Type Diabodies

Diabodies possessing the VH Domain of CD3 mAb 1 M18 were selected forfurther characterization and comparison to diabodies possessing the VHDomain of CD3 mAb 1, CD3 mAb 1 M1 and CD3 mAb 1 M2. Thus, theDART-B-type diabodies of Table 9 were prepared comprising a DiseaseAntigen (DA) Binding Domain binding the Cancer Antigen CD123, 5T4, orCD19. The amino acid sequences of each chain are provided in detailherein (see First-Nineteenth Illustrative DART-B-type Diabodies, supra).Briefly, the diabodies were expressed in CHO cells (transient or stablytransfected) and purified over a Protein A affinity resin (e.g.,MabSelect) followed by HPLC size exclusion chromatography.

Example 4 Ability of DART-B-Type Diabodies to Bind to Disease Antigens

The ability of such diabodies to bind to their respective DiseaseAntigen (i.e., CD123 or 5T4) on the surface of MOLM-13 leukemia (CD123)or A-498 kidney carcinoma (5T4) target cancer cells was assessed usingFACS. Briefly, cells were incubated with the diabody molecules (in FACSbuffer containing 10% human AB serum) in microtiter plates. The cellswere washed and incubated with biotin-conjugated mouse anti-EK-coilantibody that recognizes the E-coil/K-coil (EK) Heterodimer-PromotingDomain of the diabodies mixed with streptavidin-phycoerythrin.Representative data of such assays are shown in FIGS. 9A-9B. The datashows that the representative diabodies were capable of binding to theirrespective Disease Antigens.

Example 5 Ability of DART-B-Type Diabodies to Bind to CD4⁺ and CD8⁺T-Cells

The ability of the CD123-binding diabodies: CD123-WT, CD123-M1, CD123-M2and CD123-M18 to bind to CD4⁺ and CD8⁺ T-cells was also assessed usingFACS. A 4420×CD3 fluorescein-binding DART-A-type diabody having the CD3Binding Domain of CD3 mAb 1 was employed as a control for CD3 binding(“4420-CD3”). Briefly, CD4+ and CD8+ T-cells were incubated with thediabody molecules (in FACS buffer containing 10% human AB serum) inmicrotiter plates. The cells were washed and incubated with a labeledanti-human Fc secondary antibody. The cells were then washed andresuspended with FACS buffer, and analyzed by flow cytometry.Representative data of such assays are shown in FIG. 10A (binding toCD8⁺ T-cells) and FIG. 10B (binding to CD4⁺ T-cells). T-CD123-M1 andT-CD123-M18 exhibit reduced binding to CD3 expressing CD4⁺ and CD8⁺T-cells.

The ability of the CD123-binding diabodies: CD123-WT, CD123-M1, CD123-M2and CD123-M18 to bind to human CD3 and CD123 was also evaluated usingBIAcore®. Briefly, diabodies at concentrations of 62.5-1000 nM werepassed over soluble human CD3 that had been immobilized to a surface(normalized; 1:1 Binding Fit). The high analyte concentrations(62.5-1000 nM) were used in order to allow evaluation of parameters forweak CD3 interactions, however, high concentrations of analyte can beassociated with a contribution of non-specific binding. In separatestudies diabodies at concentrations of 62.5-100 nM were passed oversoluble CD123 that had been His-tagged and captured to an anti-PentaHissurface (normalized; 1:1 Binding Fit). Table 14 presents the calculatedk_(a), k_(d) and KD from these studies.

TABLE 14 Binding to human CD3 Binding to human CD123 CD3 k_(a) k_(d) KDk_(a) k_(d) KD Variant (×10⁴) (×10⁻³) (nM) (×10⁵) (×10⁻⁴) (nM) CD123-WT9.1 6.1 67 2.7 5.3 2.0 CD123-M1 9.5 80 842 3.2 3.9 1.2 CD123-M2 3.8 41108 4.2 4.5 1.1 CD123-M18 8.6 51 593 2.4 5.4 2.3

The ability of the 5T4-binding diabodies: 5T4-WT, 5T4-M1, 5T4-M2 and5T4-M18 to bind to CD3 was also evaluated using BIAcore®, as describedabove. Table 15A presents the calculated k_(a), k_(d) and KD.

TABLE 15A Binding to human CD3 k_(a) (×10⁵) k_(d) (×10⁻³) KD CD3 Variant(M⁻¹s⁻¹) (s⁻¹) (nM) 5T4-WT 1.5 5.4 36 5T4-M1 0.95 31 326 5T4-M2 3.5 41118 5T4-M18 0.75 34 453

In additional studies the ability of CD123-binding diabodies: CD123-WT,CD123-M13, CD123-M17 and CD123-M19 to bind to human CD3 and cynomolgusCD3 was also evaluated using BIAcore®. Briefly, diabodies atconcentrations of 6.25-400 nM were passed over immobilized human CD3 orcyno CD3 (1:1 Langmuir Binding Fit). Table 15B presents the calculatedk_(a), k_(d) and KD.

TABLE 15B Binding to human CD3 Binding to cyno CD3 CD3 k_(a) k_(d) KDk_(a) k_(d) KD Variant (×10⁴) (×10⁻³) (nM) (×10⁴) (×10⁻³) (nM) CD123-WT9.3 4.8 52 11 4.3 39 CD123-M13 5.9 27 458 5 29 580 CD123-M17 19 55 29017 56 329 CD123-M19 22 48 218 22 50 227

Example 6 Ability of Exemplary DART-B-Type Diabodies to MediateRedirected Cell Killing

Exemplary DART-B-type diabodies were evaluated for their ability tomediate redirected cell killing. Where indicated, HIV-WT or HIV-M18(described above) are used here as a negative control as they do notbind a Cancer Antigen. It will be understood that the HIV-WT and HIV-M18diabodies will bind cells expressing the epitope bound by the A32antibody (HIV env) on their cell surface (e.g., HIV infected cells), seefor example: WO 2014/1599401 and WO 2016/054101, and are capable ofmediating redirected cell killing of such cells.

The results of representative studies of redirected cell killingmediated by exemplary CD123×CD3 DART B-type diabody constructs arepresented in FIGS. 11A-11Q, FIGS. 12A-12E, and FIGS. 26A-26E. Theresults of representative studies of redirected cell killing mediated byexemplary 5T4×CD3 DART B-type diabody constructs are presented in FIGS.13A-13Q. The results of representative studies of redirected cellkilling mediated by exemplary CD19×CD3 DART B-type diabody constructsare presented in FIGS. 14A-14J. These assays were performed essentiallyas described above using the indicated effector and target cells,effector:target cell ratios, and incubation times described below (alsosee, FIGS. 11A, 12A, 13A and 14A). Where indicated, the release ofIFN-γ, TNF-α, IL-6, and IL-2 cytokines was determined at the end of theCTL assay using standard commercial reagents.

FIGS. 11A-11Q show the results of representative studies of redirectedcell killing mediated by CD123×CD3 DART B-type diabody constructs(possessing Fc Domains) CD123-WT, CD123-M2 and CD123-M18 using Pan-Teffector cells and MOLM-13 acute monocytic leukemia (AML) target cells(E:T=5:1, 24 h). Percent cytotoxicity is plotted in FIG. 11A. Cytokineresponses and cytotoxicity are plotted in FIGS. 11B-11Q (FIGS. 11B-11E:IFN-gamma; FIGS. 11F-11I: TNF-alpha; FIGS. 11J-11M: IL-6; FIGS. 11N-11Q:IL-2). FIGS. 11B, 11F, 11J and 11N: CD123-WT; FIGS. 11C, 11G, 11K and11O: CD123-M2; FIGS. 11D, 11H, 11L and 11P: CD123-M18; FIGS. 11E, 11I,11M and 11Q: HIV-WT (Negative Control). Similar cytotoxicity wasobserved against another AML cell line, MV-4-11.

FIG. 11A shows that CD123×CD3 Binding Molecules comprising different CD3mAb 1 variants exhibited markedly differing abilities to mediatecytotoxicity particularly as measured by comparing EC₅₀, but reaching asimilar maximum cytotoxicity. In addition, these molecules exhibiteddiffering abilities to mediate cytokine responses. For example, as seenin FIGS. 11B-11Q, although CD123-M18 exhibited levels of maximalcytotoxicity that were similar to those exhibited by CD123-WT, thelevels of cytokines IFN-α, TNF-α and IL-6 released by treatment withCD123-M18 were approximately 50% of the levels released by treatmentwith CD123-WT, and the level of IL-2 observed with CD123-M18 wassignificantly less than the IL-2 level observed with CD123-WT. Thus,CD123-M18 was found to be able to provide a therapeutic value that wascomparable to that of CD123-WT, but with less attending side effectsthan CD123-WT.

FIGS. 12A-12E show the results of representative studies of redirectedcell killing mediated by CD123×CD3 DART B-type diabody constructs(possessing Fc Domains) using PBMC effector cells and MOLM-13 AML targetcells. Percent cytotoxicity is plotted in FIG. 12A (E:T=15:1, 24 h).Cytokine responses (measured in a milliplex cytokine assay) are plottedin FIGS. 12B-12E (FIG. 12B: IFN-gamma; FIG. 12C: TNF-alpha; FIG. 12D:IL-6; FIG. 12E: IL-2).

The results again demonstrate that CD123-WT and CD123-M18 exhibitsimilar levels of maximal cytotoxicity, but CD123-M18 exhibited markedlyreduced cytokine responses. Additionally, the EC₅₀ values of CD123-M18for release of IFN-γ, TNF-α or IL-6 were substantially more those ofCD123-WT indicating that treatment with CD123-M18 resulted insignificantly less cytokine release as compared to treatment withCD123-WT.

FIGS. 26A-26D and FIGS. 27A-27D show the results of studies ofredirected cell killing mediated by CD123×CD3 DART B-type diabodyconstructs (possessing Fc Domains) CD123-WT, CD123-M1, CD123-M13,CD123-M17, CD123-M18 and CD123-M19 using Pan-T effector cells andMOLM-13 acute monocytic leukemia (AML) target cells (E:T=5:1, 48 to 96hours). As noted below, DART-A-WT was included as a comparator in somestudies. FIGS. 26A-26D show the results of a representative studyperformed for 48 hours. In FIG. 26A cytotoxicity is plotted, as afunction of % LDH released. Cytokine responses and cytotoxicity areplotted in FIGS. 26B-26E (FIG. 26B: IFN-gamma; FIG. 26C: TNF-alpha; FIG.26D: IL-6; FIG. 26E: IL-2). FIGS. 27A-27D summarize the results from 4-7such studies performed for 48 and 96 hours that included DART-A-WT.FIGS. 27A-27C provide comparative plots of the cytotoxicity (CTL)activity at 48 and 96 hours from four such studies (FIG. 27A: CTLactivity EC₅₀ values in pM; FIG. 27B: CTL activity as a multiple of theEC₅₀ value of CD123-WT, FIG. 27C: CTL activity Emax as a percent ofCD123-WT). FIG. 27D plots the Therapeutic Index for CTL Activity againstthe cytokine IL-2.

FIG. 26A shows that while CD123×CD3 Binding Molecules comprisingdifferent CD3 mAb 1 variants exhibited markedly different cytotoxicitycurves with CD123-M13, CD123-M17, CD123-M18 and CD123-M19, they are ableto reach a similar maximum cytotoxicity. As was seen above, each of thevariants mediated lower cytokine responses. For example, as seen inFIGS. 26B-26D, although CD123-M13, CD123-M17, CD123-M18 and CD123-M19exhibited levels of maximal cytotoxicity that were similar to thoseexhibited by CD123-WT, the levels of cytokines IFN-α, TNF-α, IL-6 andIL-2 released were significantly less than the level observed withCD123-WT treatment. Thus, each of the diabody molecules comprising theCD3 mAb 1 variants M13, M17, M18 and M19 were found to be able toprovide a therapeutic value that was comparable to that of diabodyconstructs comprising wild-type CD3 mAb 1, but with less attending sideeffects.

The results shown in FIG. 27A-27C further demonstrate that CD123-M13,CD123-M17, CD123-M18 and CD123-M19 exhibit marked different cytotoxicityEC₅₀ values but reach a maximum CTL activity that is comparable toCD123-WT and DART-A-WT. Using IL-2 as a representative cytokine, aTherapeutic Index (TI) was determined as follows:

TI=E _(max)(CTL):E _(max)(cytokine)

The calculated TI values normalized to the values for CD123-WT areplotted in FIG. 27D and further demonstrate that DA×CD3 BindingMolecules comprising the CD3 mAb 1 variants M13, M17, M18 and M19exhibit an enhanced TI over those comprising wild-type CD3 mAb 1.

FIGS. 13A-13Q show the results of representative studies of redirectedcell killing mediated by 5T4×CD3 DART B-type diabody constructs(possessing Fc Domains,) 5T4-WT, 5T4-M1, 5T4-M2, and 5T4-M18, usingPan-T effector cells and A498 renal cell carcinoma target cells(E:T=5:1, 24 h). Percent cytotoxicity is plotted in FIG. 13A. Cytokineresponses and cytotoxicity are plotted in FIGS. 13B-13Q (FIGS. 13B-13E:IFN-gamma; FIGS. 13F-13I: TNF-alpha; FIGS. 13J-13M: IL-6; FIGS. 13N-13Q:IL-2). FIGS. 13B, 13F, 13J and 13N: 5T4-WT; FIGS. 13C, 13G, 13K and 13O:5T4-M2; FIGS. 13D, 13H, 13L and 13P: 5T4-M18; FIGS. 13E, 13I, 13M and13Q: HIV-WT (Negative Control). Cytotoxicity was also observed againstJIMT-1 breast carcinoma cells. These results demonstrate that 5T4-WT and5T4-M18 exhibit similar levels of maximal cytotoxicity, but markedlydifferent cytokine responses, with 5T4-M18 exhibiting significantlyreduced levels of cytokine release as compared to 5T4-WT.

FIGS. 14A-14J show the results of representative studies of redirectedcell killing mediated by CD19×CD3 DART B-type diabody constructs(possessing Fc Domains), CD19-WT, and CD19.1-M18, using Pan-T, or PBMCeffector cells and Raji lymphoblastoid target cells (E:T=30:1 for PBMCsand 10:1 for Pan-T-cells, 24-48 h). Percent cytotoxicity (48 hrs) isplotted in FIG. 14A (PBMCs) and FIG. 14F (Pan-T-cells). Cytokineresponses at 48 hours using PBMCs are plotted in FIGS. 14B-14E (PBMCs)and FIGS. 14G-14J (Pan T-cells) (FIGS. 14B and 14G: IFN-gamma; FIGS. 14Cand 14H: TNF-alpha; FIGS. 14D and 14I: IL-6; FIGS. 14E and 14J: IL-2;HIV-M18 (Negative control)). CD19.1-M18 exhibited similar cytotoxicityand reduced cytokine release against Daudi target cells. These resultsdemonstrate that CD19-WT and CD19.1-M18 exhibit similar levels ofmaximal cytotoxicity, but markedly different cytokine responses withCD19.1-M18 exhibiting significantly reduced levels of cytokine releaseas compared to CD19-WT.

The results of the above studies confirm that constructs comprising theCD3 mAb 1 M18 variant exhibited higher cytotoxic (CTL) activity in CTLassays than those comprising the M1 and M2 variants. The CTL studiesalso indicate that constructs comprising the M18 variant exhibited lowercytokine responses as compared to WT, and similar to or only slightlyabove those exhibited by the less active M2 variant. Thus, the M18variant appears to have a larger window for CTL active vs cytokinerelease.

Example 7 Ability of Exemplary DART-B-Type Diabodies to Mediate T-CellActivation

The ability to mediate T-cell activation measured by evaluating theability of the diabodies to affect expression of CD25 and CD69, whichare markers of T-cell activation, on CD4⁺ and CD8⁺ T-cell populations.The T-cell populations were obtained from CTL assays, which wereperformed essentially as described above. Where indicated, CD4⁺ and CD8⁺T lymphocyte populations were assessed for up-regulation of theactivation markers CD69 and CD25 by flow cytometry at the end of the CTLassay.

Representative data for CD123×CD3 DART-B-type diabody constructs isshown in FIGS. 15A-15E. Cytotoxicity is plotted in FIG. 15A. Activationof CD4⁺ T-cells as determined by measuring CD25 is plotted in FIG. 15B.Activation of CD4⁺ T-cells as determined by measuring CD69 is plotted inFIG. 15C. Activation of CD8⁺ T-cells as determined by measuring CD25 isplotted in FIG. 15D. Activation of CD8⁺ T-cells as determined bymeasuring CD69 is plotted in FIG. 15E.

Representative data for 5T4×CD3 DART-B-type diabody constructs is shownin FIGS. 16A-16E. Cytotoxicity is plotted in FIG. 16A. Activation ofCD4⁺ T-cells as determined by measuring CD25 is plotted in FIG. 16B.Activation of CD4⁺ T-cells as determined by measuring CD69 is plotted inFIG. 16C. Activation of CD8⁺ T-cells as determined by measuring CD25 isplotted in FIG. 16D. Activation of CD8⁺ T-cells as determined bymeasuring CD69 is plotted in FIG. 16E.

The results of these studies show that constructs comprising the CD3-M18variant exhibited enhanced T-cell activation activity relative toconstructs comprising the M1 and M2 variants.

Example 8 In Vivo Activity of Exemplary DART-B-Type Diabodies in MurineModels

The in vivo activity of the CD123×CD3 DART-B-type diabodies CD123-WT andCD123-M18 were evaluated in a co-mix KG1A cell AML model (E:T=1:5).Briefly, NOD/SCID mice (6 per group) were injected with KG1A (AML) cellsco-mixed with activated human CD4+ or CD8+ T-cells (E:T=1:5) on Day 0.Vehicle control, CD123-WT (50 μg/kg), or CD123-M18 (5 μg/kg or 50 μg/kg)were subsequently administered. Tumor volume was monitored over thecourse of the study.

The results of this study are provided in FIG. 17A (CD4⁺ T-cells) andFIG. 17B (CD8⁺ T-cells). The results show that constructs comprising theM18 variant exhibited anti-tumor activity comparable to that ofconstructs comprising the WT CD3 Binding Domains.

A further study to evaluate the in vivo activity of the CD123×CD3DART-B-type diabodies was performed using a reconstituted tumor model inwhich 5×10⁶ KG1A (AML) cells were subcutaneously (SC) injected intoMHCI^(−/−) mice (5 female per group) on Day 0. On Day 9 1×10⁷ PBMC cellwere injected intraperitoneal (IP). Vehicle control, CD123-WT, CD123-M2or CD123-M18 (each at 0.5, 5, 50, or 500 μg/kg) were administeredintravenously (IV) twice a week (2QW) starting on Day 15. Tumor volumewas monitored over the course of the study.

The results of this study are provided in FIGS. 18A-18D. The resultsshow that CD123-M2 exhibited no activity in this model (FIG. 18B). Incontrast, CD123-M18 (FIG. 18C) exhibited anti-tumor activity comparableto that of CD123-WT (FIG. 18A) particularly at 50 μg/kg and 500 μg/kgdoses (FIG. 18D).

Another study to evaluate the in vivo activity of the CD123×CD3DART-B-type diabodies was performed using a PBMC engraftment model inwhich 5×10⁶ MV4-11 (leukemia) cells were injected SC and 1×10⁷ PBMC cellwere injected retro-orbitally (RO) into MHCI^(−/−) mice (6 males pergroup) on Day 0. Vehicle control, CD123-WT (at 0.5, 5, 50, or 500μg/kg), CD123-M18 or CD123-M2 (each at 5, 50, 500 or 1000 μg/kg) wereadministered intravenously (IV) twice a week (2QW) starting on Day 14.Tumor volume was monitored over the course of the study.

The results of this study are provided in FIGS. 19A-19D. The resultsshow that CD123-WT exhibited anti-tumor activity at 0.5 μg/kg and above(FIG. 19A). CD123-M18 exhibited anti-tumor activity at 50 μg/kg andabove (FIG. 19B). In contrast, CD123-M2 only exhibited anti-tumoractivity at 1000 μg/kg (FIG. 19C). As shown in FIG. 19D, CD123-M18anti-tumor activity is comparable to that of CD123-WT at 500 g/kg, whileCD123-M2 exhibited little or no anti-tumor activity at thisconcentration.

The in vivo activity of the 5T4×CD3 DART-B-type diabodies, 5T4-WT,5T4-M1 and 5T4-M18, were evaluated in a in PBMC engraftment model inwhich 5×10⁶ SKOV3 (ovarian carcinoma) cells were injected SC and 1×10⁷PBMC cell were injected RO into MHCI^(−/−) mice (8 females per group) onDay 0. Vehicle control, 5T4-WT (10, 50, 100, or 500 μg/kg), 5T4-M18 (10,50, 100, or 500 μg/kg), or 5T4-M2 (500 μg/kg) were administeredintravenously (IV) twice a week (2QW) starting on Day 7. Tumor volumewas monitored over the course of the study.

The results of this study are provided in FIGS. 20A-20B. The resultsshow that 5T4-WT exhibited potent anti-tumor activity at all dosestested (FIG. 20A). 5T4-M18 exhibited dose dependent anti-tumor activitythat was comparable to 5T4-WT at 500 ag/kg, while 5T4-M2 exhibitedsignificantly lower activity at 500 μg/kg (FIG. 20B).

The in vivo cytokine release profile induced by CD123×CD3 DART-B-typediabodies was examined in a PBMC co-mix model. Briefly, 5×10⁶ KG1A (AML)and 1×10⁷ PBMC cells were mixed and incubated overnight, the next daythe mixed cells were injected SC into NSG mice (6 males per group) and asingle dose of vehicle control, CD123-WT, CD123-M18 or CD123-M2 (each at50, or 500 μg/kg) was administered intravenously (IV). Six hours postadministration serum cytokine levels were evaluated. The cytokinerelease profiles are plotted in FIGS. 20A-20D (FIG. 21A: IFN-γ; FIG.21B: TNF-α; FIG. 21C: IL-6; and FIG. 21D: IL-2). The results of thesestudies show that treatment with DA×CD3 Binding Molecules comprising thevariant VL and VH Domains of CD3 mAb 1 M2, and CD3 mAb 1 M18 exhibitlower levels of cytokine release

Two further studies to evaluate the in vivo activity of the CD123×CD3DART-B-type diabodies were performed using a reconstituted tumor modelin which PBMC reconstituted (8×10⁶ PBMC injected retro-orbitally on Day0) NSG/MHCI^(−/−) mice (7-8 mice per group), were subcutaneously (SC)injected with 5×10⁶ KG1A (AML) cells on day 7. In one study, CD123-WT(0.5 mg/kg), CD123-M18 and CD123-M13 (at 0.005, 0.05, 0.5 and 1 mg/kg)or vehicle were administered intravenously (IV) twice a week (2QW)starting on Day 28. In the other study CD123-WT (0.05 mg/kg), CD123-M18and CD123-M17 (at 0.005, 0.05, 0.5 and 1 mg/kg) or vehicle wereadministered intravenously (IV) twice a week (2QW) starting on Day 28.Tumor volume was monitored over the course of the study.

The results of these studies are provided in FIGS. 28A-28B (treatmentwith CD123-WT, CD123-M18 and CD123-M13) and FIGS. 29A-29B (treatmentwith CD123-WT, CD123-M18 and CD123-M17) The results show that CD123-M18anti-tumor activity was similar to that of CD123-WT at doses of 0.5mg/kg and above (FIGS. 28A and 29A) and that CD123-M13 and CD123-M17exhibited anti-tumor activity similar to that of CD123-WT starting atjust 0.05 mg/kg, a 10 fold lower dose that for CD123-M18 (FIGS. 28B and29B).

The in vivo cytokine release profiles induced by CD123×CD3 DART-B-typediabodies CD123-WT, CD123-M13, CD123-M17 and CD123-M18 were examined ina PBMC co-mix model. Briefly, 5×10⁶ KG1A (AML) and 1×10⁷ PBMC cells weremixed and incubated overnight, the next day the mixed cells wereinjected SC into NSG mice (7-8 per group) and a single dose of CD123-WT(0.5 mg/kg), CD123-M13, CD123-M17 and CD123-M18 (at 0.05, 0.5 and 1mg/kg), or vehicle was administered intravenously (IV). Six hours postadministration serum cytokine levels were evaluated. In one studyanimals were treated with CD123-WT, CD123-M18 and CD123-M13, and in aseparate study, animals were treated with CD123-WT, CD123-M18 andCD123-M17. The IL-2 cytokine release profiles are plotted in FIGS.30A-30B (FIG. 30A: CD123-WT, CD123-M18 and CD123-M13); FIG. 30B:CD123-WT, CD123-M18 and CD123-M17) and show that treatment with DA×CD3Binding Molecules comprising the variant CD3 mAb 1 VL and VH Domainsresults in reduced levels of cytokine release as compared to the DA×CD3Binding Molecule comprising the wild type VL and VH domains.

The results of these animal studies show that administration of theDA×CD3 Binding Molecules comprising the variant VL and VH Domains of CD3mAb 1, particularly the VL and VH Domains of CD3 mAb 1 M2, CD3 mAb 1M13, CD3 mAb M17, and CD3 mAb 1 M18 (i.e., vCD3-Binding Domains) resultsin reduced levels of cytokine release as compared to DA×CD3 BindingMolecules comprising the VL and VH Domains of CD3 mAb 1 (i.e.,rCD3-Binding Domain). In particular, the results of these studiesdemonstrate that DA×CD3 Binding Molecules comprising the variant VL andVH Domains of CD3 mAb 1 M13, CD3 mAb M17, and CD3 mAb 1 M18 exhibitlower levels of cytokine release while retaining anti-tumor activity invivo.

Example 9 Generation of TRIVALENT-Type Molecules

The VH and VL Domains of CD3 mAb 1, CD3 mAb 1 M1, CD3 mAb 1 M2, or CD3mAb 1 M18 were used to generate TRIVALENT-type molecules comprising aDisease Antigen (DA) Binding Domain binding the Cancer Antigen CD123 anda Binding Domain binding the effector cell antigen CD8 (“DA×CD3×CD8TRIVALENT-type molecule”). Table 10 summarizes the CD3 Binding Domainsand SEQ ID NOs. for each polypeptide chain. The amino acid sequences ofeach chain are provided in detail herein (see First-Fourth IllustrativeTRIVALENT-type molecules, supra).

Example 10

Characterization of DA×CD3×CD8 TRIVALENT-Type Molecules

The ability of T-CD123-WT, T-CD123-M1, T-CD123-M2 and T-CD123-M18 tobind CD123 expressed on MOLM-13 cells was evaluated essentially asdescribed above. In addition, the ability of these molecules to bindCD4⁺ T-cells and CD8⁺ T-cells was evaluated essentially as describedabove. The DART-B-type diabody CD123-WT is included in these studies forcomparison. Representative data from these studies is provided in FIG.22A (binding to MOLM-13 cells), FIG. 22B (binding to CD4⁺ T-cells) andFIG. 22C (binding to CD8⁺ T-cells). All of the tested molecules exhibitcomparable binding to CD123 expressing MOLM-13 cells and CD8 expressingCD8⁺ T-cells. T-CD123-M1 and T-CD123-M18 exhibit significantly reducedbinding to CD3 expressing CD4⁺ T-cells as measured by MFI (geo mean).Binding to CD8⁺ T-cells is mediated by both the CD3- and CD8-BindingDomains present in the TRIVALENT-type molecules.

The ability of T-CD123-WT, T-CD123-M1, T-CD123-M2 and T-CD123-M18 tomediate redirected cell killing was evaluated. Briefly, theTRIVALENT-type molecules were incubated in the presence of Pan-T-cell orpurified CD4⁺ or CD8⁺ T-cell effector cells and MOLM-13 target tumorcells at an effector:target cell ratio of 1:1 for 48 hours. Thecytotoxicity was determined by measuring the release of lactatedehydrogenase (LDH) into the media by damaged cells (e.g., using theCytoTox 96® Non-Radioactive Cytotoxicity Assay Kit (Promega) thatquantitatively measures LDH release, or similar). Cytokine response wasexamined in the Pan-T-cell samples. The DART-B-type diabody CD123-WT isincluded in these studies for comparison. Percent cytotoxicity isplotted in in FIG. 23A-23C (FIG. 23A: Pan-T-cells; FIG. 23B: CD4⁺T-cells; and FIG. 23C: CD8⁺ T-cells). Cytokine responses are plotted inFIGS. 23D-23G (FIG. 23D: IFN-gamma;

FIG. 23E: TNF-alpha; FIG. 23F: IL-6; FIG. 23G: IL-2).

Example 11

Toxicology Studies

The safety and cytokine release profiles of representative CD123×CD3Binding Molecules was assessed in a dosing study in cynomolgus monkeys.In this study the potential toxicity and cytokine release profiles ofCD123-M18 (comprising the vCD3-Binding Domain of CD3 mAb 1 M18) andCD123-WT (comprising the rCD3-Binding Domain of CD3 mAb 1), whenadministered by repeated intravenous infusions was evaluated. Cellkilling activity is not readily accessed in this model. The study designis presented in Table 16.

TABLE 16 Group Dose Level Dosing No. of Animals No. Test Material(mg/kg) Days (male) 1 Control 0 0 2 2 CD123-WT 0.003 0, 7 3 3 CD123-M1810 0, 7 3 4 CD123-M18 20 0, 7 2

No mortality, body weight loss or other adverse observations wereobserved in the CD123-M18 treatment groups (10 and 20 mg/kg). Inaddition, no significant hematology or clinical chemistry changes wereobserved for in these groups. In contrast, the CD123-WT molecule (0.003mg/kg) was not well tolerated. Cytokine release syndrome and mortality(1/3) was observed in this group. The serum cytokine levels (Days 0-9)are plotted in FIGS. 24A-24E (FIG. 24A: IFN-γ, FIG. 24B: TNF-α, FIG.24C: IL-6, FIG. 24D: IL-2, and FIG. 24E: IL-15). In addition, T-cellproliferation was examined by FACS using expression of Ki67 as a markerof proliferating cells. FIG. 24F plots CD4+ T-cell expansion, and FIG.24G plots CD8⁺ T-cells expansion as a percent of CD4⁺ or CD8⁺ cellspositive for Ki67 (Days 0-14). FIGS. 24H-24I present plots of severalhematology and clinical chemistry markers of significance for theanimals in the treatment groups (FIG. 24H: Platelet counts; FIG. 24I:C-reactive protein; FIG. 24J: Urea Nitrogen). The results of this studyshow that DA×CD3 Binding Molecules comprising the vCD3-Binding DomainCD3 mAb 1 M18 are well tolerated in cynomolgus monkeys and exhibitminimal, transient increases in release of TNF-α, IFN-γ, IL-2 and IL-6even at doses exceeding projected therapeutic levels, and exhibitsmaller changes in multiple clinical chemistry markers. In addition,DA×CD3 Binding Molecules comprising the vCD3-Binding Domain CD3 mAb 1M18 were seen to preferentially stimulate proliferation of CD8⁺ T-cells.

A further toxicology study was performed with CD123-M13 (comprising thevCD3-Binding Domain of CD3 mAb 1 M13) dosed at 1 mg/kg and 10 mg/kg,CD123-M17 (comprising the vCD3-Binding Domain of CD3 mAb 1 M17) dosed at1 mg/kg and 10 mg/kg, and CD123-M19 (comprising the vCD3-Binding Domainof CD3 mAb 1 M19) dosed at 10 mg/kg. In these studies, CD123-M13 wasobserved to exhibited higher cytokine release than CD123-M17 orCD123-M19, particularly in the 10 mg/kg group. Some mortality wasobserved in this study, particularly in the CD123-M13 high dose groupand transient hematological and clinical chemistry changes wereobserved. Table 17 provides a summary of observed mortality from thisstudy and previous toxicology studies with CD123-WT and CD123-M19.

TABLE 17 CD3 Variant Dose # Dosed # Died CD123-WT 10 μg/kg 1 1Euthanasia on Day 3 3 → 3 μg/kg 3 1 Euthanasia on Day 8 (Day 1 → 8) 3 →10 μg/kg 2 1 Euthanasia on Day 8 (Day 1 → 8) 3 → 30 μg/kg 2 2 Euthanasiaon Day 8 (Day 1 → 8) or 9 CD123-M13 1 mg/kg 2 1 Euthanasia on Day 3 10mg/kg 2 2 Death or euthanasia on Day 3 CD123-M17 1 mg/kg 2 0 Nomortality 10 mg/kg 5 1 Euthanasia on Day 4 CD123-M18 No mortality at 10(n = 3) or 20 mg/kg (n = 2) CD123-M19 No mortality at 10 mg/kg (n = 2)(higher doses not tested)

As shown in Table 17 mortality is observed in animals treated with aslittle as 3 μg/kg (0.003 mg/kg) CD123-M13 (comprising the rCD3-BindingDomain of CD3 mAb 1) while CD123×CD3 Binding Molecules comprising thevCD3-Binding Domain of CD3 mAb 1 M13, M17, M18, and M19 are tolerable atmuch higher doses and exhibit reduced cytokine release profiles ascompared to CD123-M13 comprising the rCD3-Binding Domain of CD3 mAb 1.The tolerated dose ranking from these studies isCD123-M18>CD123-M19>CD123-M17>CD123-M13>CD123-WT. These findings trackwith the Therapeutic Index evaluation provided above.

Example 12

Ability of Exemplary CD123×CD3 Molecules to Mediate AML Blast Depletion

Exemplary CD123×CD3 diabodies were evaluated for their ability tomediate AML blast cell depletion from peripheral blood samples from anAML patient. Briefly, peripheral blood cells from an AML patient wereincubated in supplemented medium in the presence of increasingconcentrations of DART-A-WT, CD123-WT, CD123-M1 and CD123-M18.Cellularity (CD34⁺ blasts, CD3⁺ and CD8⁺ T-cells) were analyzed by flowcytometry at time 0 and on day 6 and is plotted as a percent ofuntreated control or as fold increase of baseline. Cytokine levels wereanalyzed by cytokine-bead array (BD) on supernatants harvested on day 4of incubation. The results of this study are presented in FIGS. 25A-25G.As shown in FIG. 25A, CD123-M18 was able to mediate depletion AML blastcells to the same extent as DART-A-WT and CD123-WT. However, CD123-M18exhibited significantly reduced expansion of T-cell population (FIG.25B: CD4⁺ T-cells; FIG. 25C: CD8⁺ T-cells). Furthermore, CD123-M18exhibited significantly lower levels of cytokine release (FIG. 25D:IFN-γ; FIG. 25E: TNF-α; FIG. 25F: IL-6; and FIG. 25G: IL-2).

These results further demonstrate that DA×CD3 Binding Moleculescomprising the vCD3-Binding Domains of CD3 mAb 1 M18 retained maximumkilling potential with slightly reduced potency, but commensurablygreater reduction in target-induced cytokine release in vitro and invivo. Incorporating such vCD3-Binding Domains into DA×CD3 BindingMolecules may expand the therapeutic index in redirected T-cell killingapplications.

Example 13

Ability of Exemplary CD19×CD3 Molecules to Mediate Autologous B-cellDepletion

In one set of studies, the exemplary CD19×CD3 diabodies CD19-WT (apositive control comprising the rCD3-Binding Domain of CD3 mAb 1) andCD19.1-M18 (comprising the vCD3-Binding Domain of CD3 mAb 1 M18), wereevaluated for their ability to mediate autologous B-cell depletion invitro and in vivo. For the in vitro studies PMBCs from human andcynomolgus monkey were utilized. Briefly, PMBCs isolated from human orcynomolgus monkey were incubated in supplemented medium in the presenceof increasing concentrations of CD19-WT (a positive control) orCD19.1-M18 or the negative control HIV-M18. B-cell levels were analyzedby flow cytometry (using CD20 as a B-cell marker) at 48 hours postincubation. Cytokine levels in the supernatants from the human sampleswere analyzed by cytokine-bead array (BD). The results of this study arepresented in FIGS. 31A-31F. As shown in FIGS. 31A-31B, CD19.1-M18 wasable to deplete autologous B-cells from both human and cynomolgus monkeyPMBCs to the same extent as CD19-WT. Furthermore, CD19.1-M18 exhibitedsignificantly lower levels of cytokine release (FIG. 31C: IFN-γ; FIG.31D: TNF-α; FIG. 31E: IL-6; and FIG. 31F: IL-2).

The ability of the positive control, CD19-WT and CD19.1-M18 (comprisingthe vCD3-Binding Domain of CD3 mAb 1 M18), to mediate autologous B-celldepletion in vivo was assessed in a dosing study in cynomolgus monkeys.The study design is presented in Table 18.

TABLE 18 Group Dose Level No. of Animals No. Test Material (mg/kg)(male) 1 CD19-WT 0.1 2 2 CD19.1-M18 1 2 3 CD19.1-M18 10 2 4 CD19.1-M1830 2

The CD19×CD3 diabodies were administered by a single 2-hr intravenousinfusion on Day 0. Peripheral blood samples were taken predose andperiodically postdose. B-cell levels in peripheral blood samples wereanalyzed by flow cytometry (using CD20 as a B-cell marker).Representative data from 1 of 2 monkeys treated in groups 1-3 are shownin FIGS. 32A-32D (FIG. 32A: predose Day 0; FIG. 32B: Day 1; FIG. 32C:Day 8;

FIG. 32D: Day 15; B-cell populations are indicated with an oval). Inaddition, inguinal lymph nodes collected predose, at Day 7, and at Day15 were stained for B-cells (using CD20 as a B-cell marker).Representative immunohistochemistry images from pre-dose and Day7samples from 1 of 2 monkeys treated in groups 1, 3 and 4 are shown inFIG. 33A-33C (FIG. 33A: group 1; FIG. 33B: group 3; FIG. 33C: group 4;predose on the left and Day 7 on the right; stained B-cells appeardark). The results of this in vivo study show that B-cells wereefficiently depleted in peripheral blood within one day and that thedepletion persisted for up to 15 days after administration (see FIGS.32A-32D) of a single dose of exemplary CD19×CD3 diabody CD19.1-M18.Similarly, B-cells were efficiently depleted in lymph nodes within 7days (the earliest time point examined after administration)demonstrating that CD19.1-M18 at doses of as little as 1 mg/kg iscapable of mediating autologous B-cell depletion via T-cell redirectedkilling in vivo to a similar degree as the CD19-WT positive control.

The ability of additional CD19×CD3 Binding Molecules to mediateautologous B-cell depletion was assessed in cynomolgus monkeys. In thisstudy the activity of CD19-WT (a positive control comprising therCD3-Binding Domain of CD3 mAb 1); CD19.1-M13 (comprising thevCD3-Binding Domain of CD3 mAb 1 M13); and CD19.1-M17 (comprising thevCD3-Binding Domain of CD3 mAb 1 M17) were evaluated for their abilityto mediate autologous B-cell depletion when administered by repeatedintravenous infusions. The study design is presented in Table 19.

TABLE 19 Group Dose Level Dosing No. of No. Test Material (mg/kg) DaysAnimals 1 CD19-WT 0.1 1, 8 2M 2 CD19.1-M13 1 1, 8 3M 3 CD19.1-M17 1 1, 83M

No mortality, body weight loss or other significant adverse observationswere observed, cold limbs were observed after dosing in one animal ingroup 3 on Day 1 and in one animal in group 2 on Day 8, both resolved bythe next day. B-cell levels in peripheral blood samples (taken predoseand periodically postdose) were analyzed by flow cytometry (using CD20as a B-cell marker). In addition, tissue samples (spleen, bone marrowand lymph nodes (LN)) were evaluated by immunohistochemistry for CD20staining. Flow cytometry data from groups 1-3 are shown in FIG. 34 andtissue staining data from each animal (and an untreated negative controlanimal) are summarized in Table 20. Serum cytokine levels were evaluatedover the course of the study (predose and periodically postdose). Serumlevels of TNF-α, IFN-γ, IL-2, IL-6, and IL-15 for each treatment groupare plotted in FIGS. 35A-35E, respectively. In addition, T-cellpopulations in peripheral blood were examined by FACS using expressionof Ki67 as a marker of proliferating cells. FIG. 36A plots CD4⁺ T-cellexpansion, and FIG. 36B plots CD8⁺ T-cells expansion as a percent ofCD4⁺ or CD8⁺ cells positive for Ki67 (taken predose and periodicallypostdose).

TABLE 20 CD20 Tissue Staining Neg Control‡ CD19-WT Animal Number 50011001 1002 Spleen X 2+ 1+ Bone marrow 2+ 0  0  LN, axillary 3+ 1+ 1+ LN,mandibular 3+ 1+ 1+ LN, mesenteric 3+ 1+ 1+ LN, inguinal 3+ 1+ 1+CD19.1-M13 Animal Number 2001 2002 2003 Spleen 1+ 2+ 1+ Bone marrow 0 0  0  LN, axillary 1+ 2+ 1+ LN, mandibular 1+ 1+ 1+ LN, mesenteric 0  1+1+ LN, inguinal 1+ 1+ 1+ CD19.1-M17 Animal Number 3001 3002 3003 Spleen1+ 1+ 1+ Bone marrow 0  1+ 1+ LN, axillary 1+ 2+ 2+ LN, mandibular 1+ 2+2+ LN, mesenteric 1+ 1+ 2+ LN, inguinal 1+ 2+ 2+ ‡one animal in negativecontrol group X—Not examined 0—No staining observed 1+—Weak stainingobserved 2+—Moderate staining observed 3+—Strong staining observed

The results of this study show that CD19×CD3 Binding Moleculescomprising the vCD3-Binding Domain CD3 mAb 1 M13 or CD3 mAb 1 M17 wereactive with animals treated with 1 mg/kg CD19.1-M13 exhibitingautologous B-cell depletion to a similar degree as the CD19-WT positivecontrol. Animals treated with 1 mg/kg CD19.1-M17 also exhibitedautologous B-cell depletion, but to a slightly lesser extent than thepositive control. It is expected that CD19.1-M17 would achievecomparable depletion as higher dosages. The variants mediated much lowerincreases in the release of INF-γ, TNF-α, IL-2, and IL-6 and slightreductions in the release of IL-15. In addition, binding moleculescomprising CD3 mAb 1 M13 and CD3 mAb 1 M17 were seen to stimulateproliferation of T-cells, with molecules comprising CD3 mAb 1 M13exhibiting higher levels of proliferation. In addition, both moleculesexhibited preferential stimulation of proliferation of CD8⁺ T-cells.

Together the studies provided in the above examples show that DA×CD3Binding Molecules comprising the vCD3-Binding Domain of CD3 mAb 1 (e.g.,M13, M17, M18, M19), exhibit a range of binding affinities, a range ofcytotoxicity EC₅₀ values but all reach a maximum CTL activity that iscomparable to molecules comprising the rCD3-Binding Domain of CD3 mAb 1,thus exhibit an enhanced Therapeutic Index. These studies further showthat such molecules are tolerated and active at mediating T-cellredirected cell killing, and at stimulating T-cell activation andproliferation in vivo.

All publications and patents mentioned in this specification are hereinincorporated by reference to the same extent as if each individualpublication or patent application was specifically and individuallyindicated to be incorporated by reference in its entirety. While theinvention has been described in connection with specific embodimentsthereof, it will be understood that it is capable of furthermodifications and this application is intended to cover any variations,uses, or adaptations of the invention following, in general, theprinciples of the invention and including such departures from thepresent disclosure as come within known or customary practice within theart to which the invention pertains and as may be applied to theessential features hereinbefore set forth.

1. A DA×CD3 Binding Molecule comprising a CD3-Binding Domain capable ofbinding an epitope of CD3 and a Disease Antigen-Binding Domain capableof binding an epitope of 5T4, CD19 or the env protein of HIV, whereinsaid CD3-Binding Domain comprises: (I) (A) a CDR_(H)1 Domain comprisingthe amino acid sequence of SEQ ID NO:99; (B) a CDR_(H)2 Domaincomprising the amino acid sequence of SEQ ID NO:58; (C) a CDR_(H)3Domain comprising the amino acid sequence of SEQ ID NO:59; (D) aCDR_(L)1 Domain comprising the amino acid sequence of SEQ ID NO:60; (E)a CDR_(L)2 Domain comprising the amino acid sequence of SEQ ID NO:61;and (F) a CDR_(L)3 Domain comprising the amino acid sequence of SEQ IDNO:62; or.
 2. The DA×CD3 Binding Molecule of claim 1, wherein saidCD3-Binding Domain comprises: (I) (A) a VL Domain comprising the aminoacid sequence of SEQ ID NO:56; (B) a VH Domain comprising the amino acidsequence of SEQ ID NO:98.
 3. The DA×CD3 Binding Molecule of claim 1,wherein said DA×CD3 Binding Molecule is a bispecific antibody, abispecific diabody, a bispecific scFv, a bispecific TandAb, or atrivalent binding molecule. 4-10. (canceled)
 11. The DA×CD3 BindingMolecule of claim 1, wherein said DA×CD3 Binding Molecule comprises: afirst polypeptide chain and a second polypeptide chain, covalentlybonded to one another, wherein: (A) the first polypeptide chaincomprises, in the N-terminal to C-terminal direction: (i) a Domain 1,comprising: (1) (a) sub-Domain (1A), which comprises a VL Domain of amonoclonal antibody capable of binding to said epitope of 5T4(VL_(5T4)); or (b) a sub-Domain (1A), which comprises a VL Domain of amonoclonal antibody capable of binding to said epitope of CD19(VL_(CD19)); or (c) a sub-Domain (1A), which comprises a VL Domain of amonoclonal antibody capable of binding to said epitope of the envprotein of HIV (VL_(HIV)); and (2) a sub-Domain (1B), which comprises aVH Domain of a monoclonal antibody capable of binding to said epitope ofCD3 (VH_(CD3)); wherein said sub-Domains 1A and 1B are separated fromone another by a peptide Linker; and (ii) a Domain 2, wherein saidDomain 2 is a Heterodimer-Promoting Domain; (B) the second polypeptidechain comprises, in the N-terminal to C-terminal direction: (i) a Domain1, comprising: (1) a sub-Domain (1A), which comprises a VL Domain ofsaid monoclonal antibody capable of binding to said epitope of CD3(VL_(CD3)); and (2) (a) sub-Domain (1B), which comprises a VH Domain ofsaid monoclonal antibody capable of binding to said epitope of 5T4(VH_(5T4)); or (b) a sub-Domain (1B), which comprises a VH Domain ofsaid monoclonal antibody capable of binding to said epitope of CD19(VH_(CD19)): or (c) a sub-Domain (1B), which comprises a VH Domain ofsaid monoclonal antibody capable of binding to said epitope of the envprotein of HIV (VH_(HIV)): wherein said sub-Domains 1A and 1B areseparated from one another by a peptide Linker; (ii) a Domain 2, whereinsaid Domain 2 is a Heterodimer-Promoting Domain, wherein saidHeterodimer-Promoting Domain of said first and said second polypeptidechains are different; and wherein: (a) the VL Domain of the firstpolypeptide chain and the VH Domain of the second polypeptide chainassociate to form the 5T4, CD19 or the env protein of HIV bindingdomain, and the VH Domain of the first polypeptide chain and the VLDomain of the second polypeptide chain associate to form the CD3-BindingDomain; or (b) the VL Domain of the first polypeptide chain and the VHDomain of the second polypeptide chain associate to form the CD3-BindingDomain, and the VH Domain of the first polypeptide chain and the VLDomain of the second polypeptide chain associate to form the 5T4, CD19or the env protein of HIV binding domain.
 12. The DA×CD3 BindingMolecule of claim 11, wherein: (a) said Heterodimer-Promoting Domain ofsaid first polypeptide chain is an E-coil Domain, and saidHeterodimer-Promoting Domain of said second polypeptide chain is aK-coil Domain; or (b) said Heterodimer-Promoting Domain of said firstpolypeptide chain is a K-coil Domain, and said Heterodimer-PromotingDomain of said second polypeptide chain is an E-coil Domain.
 13. TheDA×CD3 Binding Molecule of claim 11, wherein the first or secondpolypeptide chain additionally comprises a Domain 3 comprising a CH2 andCH3 Domain of an immunoglobulin Fc Domain.
 14. The DA×CD3 BindingMolecule of claim 13, wherein said DA×CD3 Binding Molecule furthercomprises a third polypeptide chain comprising a CH2 and CH3 Domain ofan immunoglobulin Fc Domain. 15-16. (canceled)
 17. A pharmaceuticalcomposition that comprises the DA×CD3 Binding Molecule of claim 1 and apharmaceutically acceptable carrier.
 18. A method for the treatment of adisease, comprising administering to a subject in need thereof atherapeutically effective amount of the pharmaceutical composition ofclaim
 17. 19-27. (canceled)
 28. The DA×CD3 Binding Molecule of claim 1,wherein said Disease Antigen-Binding Domain Comprises: (I) (A) theCDR_(H)1 Domain, CDR_(H)2 Domain, and the CDR_(H)3 Domain of SEQ IDNO:156; and (B) the CDR_(L)1 Domain, the CDR_(L)2 Domain, and theCDR_(L)3 Domain of SEQ ID NO:157; or (II) (A) the CDR_(H)1 Domain,CDR_(H)2 Domain, and the CDR_(H)3 Domain of SEQ ID NO:164; and (B) theCDR_(L)1 Domain, the CDR_(L)2 Domain, and the CDR_(L)3 Domain of SEQ IDNO:165; or (III) (A) the CDR_(H)1 Domain, CDR_(H)2 Domain, and theCDR_(H)3 Domain of SEQ ID NO:164; and (B) the CDR_(L)1 Domain, theCDR_(L)2 Domain, and the CDR_(L)3 Domain of SEQ ID NO:195; or (IV) (A)the CDR_(H)1 Domain, CDR_(H)2 Domain, and the CDR_(H)3 Domain of SEQ IDNO:168; and (B) the CDR_(L)1 Domain, the CDR_(L)2 Domain, and theCDR_(L)3 Domain of SEQ ID NO:169.
 29. The DA×CD3 Binding Molecule ofclaim 1, wherein said Disease Antigen-Binding Domain Comprises: (I) (A)a VH Domain comprising the amino acid sequence of SEQ ID NO:156; and (B)a VL Domain comprising the amino acid sequence of SEQ ID NO:157; or (II)(A) a VH Domain comprising the amino acid sequence of SEQ ID NO:164; and(B) a VL Domain comprising the amino acid sequence of SEQ ID NO:165; or(III) (A) a VH Domain comprising the amino acid sequence of SEQ IDNO:164; and (B) a VL Domain comprising the amino acid sequence of SEQ IDNO:195; or (IV) (A) a VH Domain comprising the amino acid sequence ofSEQ ID NO:168; and (B) a VL Domain comprising the amino acid sequence ofSEQ ID NO:169.
 30. The DA×CD3 Binding Molecule of claim 2, wherein saidDisease Antigen-Binding Domain Comprises: (I) (A) a VH Domain comprisingthe amino acid sequence of SEQ ID NO:156; and (B) a VL Domain comprisingthe amino acid sequence of SEQ ID NO:157; or (II) (A) a VH Domaincomprising the amino acid sequence of SEQ ID NO:164; and (B) a VL Domaincomprising the amino acid sequence of SEQ ID NO:165; or (III) (A) a VHDomain comprising the amino acid sequence of SEQ ID NO:164; and (B) a VLDomain comprising the amino acid sequence of SEQ ID NO:195; or (IV) (A)a VH Domain comprising the amino acid sequence of SEQ ID NO:168; and (B)a VL Domain comprising the amino acid sequence of SEQ ID NO:169.
 31. TheDA×CD3 Binding Molecule of claim 11, wherein: (I) (A) said VH5T4comprises the amino acid sequence of SEQ ID NO:156; and (B) said VL5T4comprises the amino acid sequence of SEQ ID NO:157; or (A) saidVH_(CD19) comprises the amino acid sequence of SEQ ID NO:164; and (B)said VL_(CD19) comprises the amino acid sequence of SEQ ID NO:165; or(A) said VH_(CD19) comprises the amino acid sequence of SEQ ID NO:164;and (B) said VL_(CD19) comprises the amino acid sequence of SEQ IDNO:195; or (A) said VH_(HIV) comprises the amino acid sequence of SEQ IDNO:168; and (B) said VL_(HIV) comprises the amino acid sequence of SEQID NO:169; and (II) (C) said VH_(CD3) comprises the amino acid sequenceof SEQ ID NO:98; and (D) said VL_(CD3) comprises the amino acid sequenceof SEQ ID NO:56.
 32. The DA×CD3 Binding Molecule of claim 12, wherein:(I) (A) said VH5T4 comprises the amino acid sequence of SEQ ID NO:156;and (B) said VL5T4 comprises the amino acid sequence of SEQ ID NO:157;or (A) said VH_(CD19) comprises the amino acid sequence of SEQ IDNO:164; and (B) said VL_(CD19) comprises the amino acid sequence of SEQID NO:165; or (A) said VH_(CD19) comprises the amino acid sequence ofSEQ ID NO:164; and (B) said VL_(CD19) comprises the amino acid sequenceof SEQ ID NO:195; or (A) said VH_(HIV) comprises the amino acid sequenceof SEQ ID NO:168; and (B) said VL_(HIV) comprises the amino acidsequence of SEQ ID NO:169; and (II) (C) said VH_(CD3) comprises theamino acid sequence of SEQ ID NO:98; and (D) said VL_(CD3) comprises theamino acid sequence of SEQ ID NO:56.
 33. The DA×CD3 Binding Molecule ofclaim 13, wherein: (I) (A) said VH5T4 comprises the amino acid sequenceof SEQ ID NO:156; and (B) said VL5T4 comprises the amino acid sequenceof SEQ ID NO:157; or (A) said VH_(CD19) comprises the amino acidsequence of SEQ ID NO:164; and (B) said VL_(CD19) comprises the aminoacid sequence of SEQ ID NO:165; or (A) said VH_(CD19) comprises theamino acid sequence of SEQ ID NO:164; and (B) said VL_(CD19) comprisesthe amino acid sequence of SEQ ID NO:195; or (A) said VH_(HIV) comprisesthe amino acid sequence of SEQ ID NO:168; and (B) said VL_(HIV)comprises the amino acid sequence of SEQ ID NO:169; and (II) (C) saidVH_(CD3) comprises the amino acid sequence of SEQ ID NO:98; and (D) saidVL_(CD3) comprises the amino acid sequence of SEQ ID NO:56.
 34. TheDA×CD3 Binding Molecule of claim 14, wherein: (I) (A) said VH5T4comprises the amino acid sequence of SEQ ID NO:156; and (B) said VL5T4comprises the amino acid sequence of SEQ ID NO:157; or (A) saidVH_(CD19) comprises the amino acid sequence of SEQ ID NO:164; and (B)said VL_(CD19) comprises the amino acid sequence of SEQ ID NO:165; or(A) said VH_(CD19) comprises the amino acid sequence of SEQ ID NO:164;and (B) said VL_(CD19) comprises the amino acid sequence of SEQ IDNO:195; or (A) said VH_(HIV) comprises the amino acid sequence of SEQ IDNO:168; and (B) said VL_(HIV) comprises the amino acid sequence of SEQID NO:169; and (II) (C) said VH_(CD3) comprises the amino acid sequenceof SEQ ID NO:98; and (D) said VL_(CD3) comprises the amino acid sequenceof SEQ ID NO:56.
 35. A DA×CD3 Binding Molecule, wherein said DA×CD3Binding Molecule comprises: (I) (A) a first polypeptide chain comprisingthe amino acid sequence of SEQ ID NO:184; (B) a second polypeptide chaincomprising the amino acid sequence of SEQ ID NO:181; and (C) a thirdpolypeptide chain comprising the amino acid sequence of SEQ ID NO:176;or (II) (A) a first polypeptide chain comprising the amino acid sequenceof SEQ ID NO:193; (B) a second polypeptide chain comprising the aminoacid sequence of SEQ ID NO:194; and (C) a third polypeptide chaincomprising the amino acid sequence of SEQ ID NO:176; or (III) (A) afirst polypeptide chain comprising the amino acid sequence of SEQ IDNO:197; (B) a second polypeptide chain comprising the amino acidsequence of SEQ ID NO:192; and (C) a third polypeptide chain comprisingthe amino acid sequence of SEQ ID NO:176; or (IV) (A) a firstpolypeptide chain comprising the amino acid sequence of SEQ ID NO:196;(B) a second polypeptide chain comprising the amino acid sequence of SEQID NO:186; and (C) a third polypeptide chain comprising the amino acidsequence of SEQ ID NO:176.
 36. A pharmaceutical composition thatcomprises the DA×CD3 Binding Molecule of claim 35 and a pharmaceuticallyacceptable carrier.
 37. A method for the treatment of a hematologicalcancer or for the treatment of HIV infection, comprising administeringto a subject in need thereof a therapeutically effective amount of thepharmaceutical composition of claim
 36. 38. The method of claim 37,wherein said hematological cancer is selected from the group consistingof: acute myeloid leukemia (AML), chronic myelogenous leukemia (CML),myelodysplastic syndrome (MDS), acute B lymphoblastic leukemia (B-ALL),chronic lymphocytic leukemia (CLL), Richter's syndrome, hairy cellleukemia (HCL), blastic plasmacytoid dendritic cell neoplasm (BPDCN),non-Hodgkin's lymphoma (NHL), including mantle cell lymphoma (MCL) andsmall lymphocytic lymphoma (SLL), Hodgkin's lymphoma, systemicmastocytosis, and Burkitt's lymphoma.
 39. The method of claim 38,wherein the pharmaceutical composition is administered intravenously.40. The method of claim 39, wherein said intravenous administration isby infusion.
 41. The method of claim 40, wherein the DA×CD3 BindingMolecule of the pharmaceutical composition is administered at a dosageof about 0.01 μg/kg to about 30 mg/kg of said subject's body weight. 42.The method of claim 40, wherein the pharmaceutical composition isadministered once a week, once every two weeks, or once a month.
 43. Ahost cell comprising: (I) (A) a polynucleotide encoding a polypeptidechain comprising the amino acid sequence of SEQ ID NO:184; (B) apolynucleotide encoding a polypeptide chain comprising the amino acidsequence of SEQ ID NO:181; and (C) a polynucleotide encoding apolypeptide chain comprising the amino acid sequence of SEQ ID NO:176;or (II) (A) a polynucleotide encoding a polypeptide chain comprising theamino acid sequence of SEQ ID NO:193; (B) a polynucleotide encoding apolypeptide chain comprising the amino acid sequence of SEQ ID NO:194;and (C) a polynucleotide encoding a polypeptide chain comprising theamino acid sequence of SEQ ID NO:176; or (III) (A) a polynucleotideencoding a polypeptide chain comprising the amino acid sequence of SEQID NO:197; (B) a polynucleotide encoding a polypeptide chain comprisingthe amino acid sequence of SEQ ID NO:192; and (C) a polynucleotideencoding a polypeptide chain comprising the amino acid sequence of SEQID NO:176; or (IV) (A) a polynucleotide encoding a polypeptide chaincomprising the amino acid sequence of SEQ ID NO:196; (B) apolynucleotide encoding a polypeptide chain comprising the amino acidsequence of SEQ ID NO:186; and (C) a polynucleotide encoding apolypeptide chain comprising the amino acid sequence of SEQ ID NO:176.